Super-enhancers and methods of use thereof

ABSTRACT

The present invention relates in some aspects to super-enhancers and related compositions, methods, and agents that are useful for modulating expression of cell type-specific genes that are required for maintenance of cell identity (e.g., embryonic stem cell identity) or maintenance of a disease state (e.g., cancer).

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application Nos.61/718,697, filed Oct. 25, 2012 and 61/799,646, filed Mar. 15, 2013. Theentire teachings of the above application(s) are incorporated herein byreference.

GOVERNMENT SUPPORT

This invention was made with government support under RO1-HG002668 andRO1-CA146445 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Regulatory elements (e.g., transcription factors, cis-acting enhancerelements, transcriptional coactivators and chromatin regulators)activate gene expression programs in cells ranging from embryonic stemcells (ESCs) to tumor cells. Regulatory elements are important formaintenance of cell identity (e.g., ESC identity) and of some diseasestates (e.g., cancer). The mechanisms underlying how regulatory elementscontribute to maintenance of cell identity and of disease state are notentirely understood.

SUMMARY OF THE INVENTION

The present invention relates in some aspects to super-enhancers andrelated compositions, methods, and agents that are useful for modulatingexpression of cell type-specific genes that are required for maintenanceof cell identity (e.g., embryonic stem cell identity) or maintenance ofa disease state (e.g., cancer).

In some aspects, the invention provides an isolated super-enhancer, orfunctional fragment and/or variant thereof, comprising a genomic regionof deoxyribonucleic acid (DNA) that contains at least two enhancers,wherein the genomic region is occupied when present within a cell bymore, e.g., 2, 3, 4, 5, 10, or 15 fold more super-enhancer component,e.g., chromatin associated protein, e.g., a transcriptional coactivator,than the average single enhancer within the cell.

A super-enhancer component, as used herein, is a component, typically aprotein, that has a higher local concentration, or exhibits a higheroccupancy, at a super-enhancer, as opposed to a normal enhancer or anenhancer outside a super-enhancer, and in embodiments, contributes toincreased expression of the associated gene.

In an embodiment the super-enhancer comprises all or part of a geneunder its control. In an embodiment does not contain a completeassociated gene.

In some embodiments the transcriptional coactivator is Mediator. In someembodiments the transcriptional coactivator is Med1.

In some embodiments the genomic region is occupied when present within acell by more super-enhancer component, e.g., more chromatin regulatorthan the average single or normal enhancer within the cell.

In some embodiments the chromatin regulator is a BET bromodomainprotein. In some embodiments the BET bromodomain protein is BRD4.

In some embodiments the genomic region spans between about 4 kilobasesand about 40 kilobases in length.

In some embodiments the genomic region spans sufficient nucleic acid, orthe super-enhancer is of sufficient size or structure, such that, whenassociated with a gene, the gene has substantially greater expressionthan in the absence of the super-enhancer.

In some embodiments the at least two enhancers are clustered together.

In some embodiments each enhancer comprises a binding site for a cognatetranscription factor.

In some embodiments the cognate transcription factor comprises anembryonic stem cell master transcription factor. In some embodiments theembryonic stem cell master transcription factor is selected from thegroup consisting of Oct4, Sox2, Nanog, Esrrb, Utf1, Klf4, mir-290-295microRNA gene cluster, Tbx3, Sgk1, and combinations thereof.

In some embodiments a super-enhancer component comprises an enzyme that,adds, detects or reads, or removes a functional group, e.g., a methyl oracetyl group, from a chromatin component, e.g., DNA or histones.

In some embodiments a super-enhancer component comprises an enzyme thatalters, reads, or detects the structure of a chromatin component, e.g.,DNA or histones, e.g., a DNA methylase or demythylase, a histonemethylase or demethylase, or a histone acetylase or de-acetylase thatwrite, read or erase histone marks, e.g., H3K4me1 or H3K27Ac.

In some embodiments a super-enhancer component comprises an enzyme,adds, detects or reads, or removes a functional group, e.g., a methyl oracetyl group, from a chromatin component, e.g., DNA or histones.

In some embodiments the super-enhancer component comprises a proteinneeded for development into, or maintenance of, a selected cellularstate or property, e.g., a state of differentiation, development ordisease, e.g., a cancerous state, or the propensity to proliferate orthe propensity or the propensity to undergo apoptosis.

In some embodiments the cognate transcription factor comprises anoncogenic transcription factors. In some embodiments the oncogenictranscription factor is selected from the group consisting of c-Myc,IRF4, p53, AP-1, Bcr-Abl, c-Fos, c-Jun and combinations thereof. In someembodiments the cognate transcription factor comprises a muscle celltranscription factor. In some embodiments the transcription factor isMyoD.

In some embodiments the cognate transcription factor comprises a B celltranscription factor. In some embodiments the transcription factor isPu.1.

In some embodiments the genomic region is occupied when present withinthe cell by an order of magnitude more super-enhancer component, e.g.,transcriptional coactivator than the average single enhancer within thecell. In some embodiments the order of magnitude is at least about2-fold. In some embodiments the order of magnitude is at least about10-fold. In some embodiments the order of magnitude is at least about15-fold. In some embodiments the order of magnitude is at least about16-fold.

In some aspects, the invention provides a composition comprising asuper-enhancer of the present invention.

In some aspects, the invention provides a nucleic acid constructcomprising a super-enhancer, or functional fragment and/or variantthereof, of the present invention. In some embodiments the nucleic acidconstruct includes a nucleotide sequence encoding a target geneoperatively linked to the super-enhancer. In some embodiments thenucleic acid construct includes a reporter construct.

In some aspects, the invention provides a cell transfected with anucleic acid construct comprising a super-enhancer, or functionalfragment and/or variant thereof, operatively linked to a target genewherein upon transfection of the cell with the nucleic acid constructendogenous transcriptional coactivators and chromatin regulators withinthe cell co-occupy the enhancers and the active transcription startsites of the target gene to stimulate high levels of expression of thetarget gene within the cell.

In some embodiments the cell is a mammalian cell. In some embodimentsthe cell is a human cell. In some embodiments the cell is an embryonicstem cell or embryonic stem cell-like cell. In some embodiments the cellis a muscle cell. In some embodiments the muscle cell is a myotube. Insome embodiments the cell is a B cell. In some embodiments the B cell isa Pro-μ cell.

In some aspects, the invention provides a method of increasing the levelof expression of a target gene in a cell, comprising transfecting a cellunder conditions suitable for expression of the target gene with anucleic acid expression construct comprising a nucleic acid sequenceencoding the target gene operatively linked to a super enhancer, orfunctional fragment and/or variant thereof, wherein upon transfection ofthe cell endogenous transcriptional coactivators and chromatinregulators within the cell co-occupy enhancers clustered within thesuper enhancer, or functional fragment and/or variant thereof, andactive transcription start sites of the target gene to increase thelevel of expression of the target gene within the cell. In someembodiments the level of expression of the target gene is increased2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or more within the cell.

In some aspects the invention provides a kit for increasing theexpression of a target gene in a cell, comprising: (a) a nucleic acidconstruct comprising an artificial super enhancer, or functionalfragment and/or variant thereof, operatively linked to the target gene;(b) a population of cells suitable for expression of said target gene;and (c) a reagent for transfecting said population of cells with saidnucleic acid construct.

In some aspects the invention provides a method of identifying a superenhancer, or functional fragment and/or variant thereof, in a cell,comprising: (a) identifying a genomic region of DNA within said cellcharacterized by a cluster of enhancers each of which bind a cognatetranscription factor capable of interacting with Mediator to stimulatetranscription of the target gene within said cell; (b) measuring in theidentified genomic region a level of Mediator; and (c) identifying thegenomic region as a super enhancer, or functional fragment and/orvariant thereof, if the level of Mediator greater than the level ofMediator occupying the average single enhancer.

In some embodiments the level of Mediator identified in the genomicregion is an order of magnitude more than the level of Mediatoroccupying the average single enhancer. In some embodiments the order ofmagnitude is at least 2-fold, at least 10-fold, at least 15-fold, atleast 16-fold, or more.

In some embodiments the super enhancer, or functional fragment and/orvariant thereof, is identified by performing chromatinimmunoprecipitation high-throughput sequencing (ChIP-Seq).

In some aspects, the invention provides a method of selectivelyinhibiting expression of an aberrantly expressed gene comprisingdisrupting the function of a super-enhancer associated with theaberrantly expressed gene.

In some embodiments the gene is an oncogene. In some embodiments theoncogene is selected from the group consisting of c-MYC and IRF4.

In some embodiments disrupting the function of the super-enhancercomprises contacting said super-enhancer region with an effective amountof an agent that interferes with occupancy of the super-enhancer regionby a cognate transcription factor for the gene, a transcriptionalcoactivator, or a chromatin regulator. In some embodiments the agent isa bromodomain inhibitor. In some embodiments the agent is a BRD4inhibitor.

In some embodiments the agent is JQ1.

In some embodiments the agent is iBET. In some embodiments the agentinterferes with a binding site on the super-enhancer for the cognatetranscription factor, interferes with interaction between the cognatetranscription factor and a transcriptional coactivator, inhibits thetranscription coactivator, or interferes with or inhibits the chromatinregulator.

In some aspects the invention provides a method of treating aproliferative disorder in a patient in need of such treatment, saidproliferative disorder characterized by an oncogene-associatedsuper-enhancer occupied by more Mediator or BRD4 than an average singleenhancer, comprising administering to the patient an effective amount ofan agent that disrupts the function of the oncogene-associatedsuper-enhancer, thereby selectively inhibiting proliferation of theoncogene in the patient.

In some embodiments the proliferative disorder is a hematologicalmalignancy.

In some embodiments the proliferative disorder is selected from thegroup consisting of acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), chronic myelogenous leukemia (CML), chroniclymphocytic leukemia (CLL), hairy cell leukemia, Hodgkin's lymphoma,non-Hodgkin's lymphoma, cutaneous T-cell lymphoma (CTCL), peripheralT-cell lymphoma (PTCL), Mantle cell lymphoma, B-cell lymphoma, acutelymphoblastic T cell leukemia (T-ALL), acute promyelocytic leukemia, andmultiple myeloma.

In some embodiments the agent is a BRD4 inhibitor. In some embodimentsthe agent is JQ1. In some embodiments the agent is iBET.

In some aspects, the invention provides a method of treating multiplemyeloma involving an IGH-MYC locus that results in aberrant expressionof oncogene c-Myc, comprising administering to a patient in need of suchtreatment an effective amount of an agent that decreases occupancylevels of BRD4 and MED1 at a super-enhancer region associated with theIGH-MYC locus, wherein decreased occupancy levels of BRD4 and MED1 atthe super-enhancer disrupt function of the super-enhancer therebydecreasing aberrant expression of oncogene c-Myc such that the multiplemyeloma is treated. In some embodiments the agent is JQ1 or iBET.

In some aspects the invention provides a method of identifying an agentthat disrupts a super-enhancer associated with a gene, e.g., asuper-enhancer identified by a method described herein, comprising:

(a) providing a cell or cell-free system having a super-enhancer, orfunctional fragment and/or variant thereof, associated with a gene,e.g., a gene which is heterologous to one or both of the cell or thesuper-enhancer, e.g., a reporter construct;

(b) contacting the cell with a test agent, e.g., under conditionssuitable for the super-enhancer, or functional fragment and/or variantthereof, to drive high levels of expression of the associated gene; and

(c) measuring the level of expression of the associated gene, e.g., areporter construct.

In an embodiment decreased expression of the associated gene in thepresence of the test agent indicates that the test agent is as an agentthat disrupts the super-enhancer associated with the gene.

In an embodiment the method comprises comparing the level of expressionwith a reference, e.g., a similar cell or cell-free system not contactedwith the test agent.

In an embodiment the method comprises confirming disruption of thesuper-enhancer, or functional fragment and/or variant thereof, e.g., byanalysis of the presence of one or more super-enhancer component.

In an embodiment the method is first performed in a cell-free system ora cell preparation, e.g., a cultured cell, and repeated in an animal.

In an embodiment the super-enhancer is associated with a gene that isexpressed in a disease state cell, e.g., a cancer cell.

The method, as well as any other method described herein, can includememorializing the results.

In some aspects the invention provides a method of identifying an agentthat disrupts a super-enhancer associated with a gene, comprising:

(a) providing a cell or cell-free system having a heterologoussuper-enhancer, or functional fragment and/or variant thereof,associated with a gene, e.g., a gene which is heterologous to one orboth of the cell or the super-enhancer, e.g., a reporter construct;

(b) contacting the cell or cell-free system with a test agent, e.g.,under conditions suitable for the super-enhancer, or functional fragmentand/or variant thereof, to drive high levels of expression of theassociated gene;

(c) and measuring the level of expression of the associated gene, e.g.,a reporter construct.

In an embodiment decreased expression of the associated gene in thepresence of the test agent indicates that the test agent is as an agentthat disrupts the super-enhancer associated with the gene.

In an embodiment the method comprises comparing the level of expressionwith a reference, e.g., a similar cell or cell-free system not contactedwith the test agent.

In an embodiment the method comprises confirming disruption of thesuper-enhancer, or functional fragment and/or variant thereof, e.g., byanalysis of the presence of one or more epigenetic super-enhancercomponent.

In an embodiment the method is first performed in a cell-free system ora cell preparation, e.g., a cultured cell, and repeated in an animal.

In an embodiment the super-enhancer is associated with a gene that isexpressed in a disease state cell, e.g., a cancer cell.

In some aspects the invention provides a method of identifying an agentthat disrupts a super-enhancer associated with a gene, comprising: (a)transfecting a cell with a super-enhancer, or functional fragment and/orvariant thereof, and the associated gene under conditions suitable forthe super-enhancer to drive high levels of expression of the associatedgene; (b) contacting the cell with a test agent; (c) and measuring thelevel of expression of the associated gene, wherein decreased expressionof the associated gene in the presence of the test agent indicates thatthe test agent is as an agent that disrupts the super-enhancerassociated with the gene.

In an embodiment the method comprises comparing the level of expressionwith a reference, e.g., a similar cell not contacted with the testagent. In an embodiment the method comprises confirming disruption ofthe super-enhancer, or functional fragment and/or variant thereof, e.g.,by analysis of the presence of one or more super-enhancer component. Inan embodiment the method is first performed in a cell-free system or acell preparation, e.g., a cultured cell, and repeated in an animal.

In an embodiment the super-enhancer is associated with a gene that isexpressed in a disease state cell, e.g., a cancer cell.

In some aspects the invention provides a method of identifying an agentthat disrupts a super-enhancer comprising: (a) transfecting a cell witha super-enhancer operably linked to a reporter construct comprising areporter gene under conditions suitable for the super-enhancer to drivehigh levels of expression of the reporter gene; (b) contacting the cellwith a test agent; (c) and measuring the level of expression of thereporter gene, wherein decreased expression of the reporter gene in thepresence of the test agent indicates that the test agent is as an agentthat disrupts the super-enhancer.

In some embodiments the super-enhancer is naturally associated with agene of interest, wherein the gene of interest is optionally adisease-associated gene, optionally an oncogene. In some embodimentsexpression is measured at least in part by measuring the level of a geneproduct encoded by the gene or by measuring activity of a gene productencoded by the gene. In some embodiments a gene product is mRNA orpolypeptide encoded by the gene.

In some aspects, the invention relates to a method of identifying asuper-enhancer, or a gene associated with a super-enhancer, comprising:

cross-linking, e.g., covalently cross-linking, chromatin, such thatchromosomal nucleic acid is cross-linked to a super-enhancer component,e.g., a chromatin associated protein, e.g., one or more of a Mediatorprotein, Med1, Oct4, Sox2, Nanog, or NOS, to form a cross-linkedcomplex;

contacting said cross-linked complex with a ligand having affinity forthe super-enhancer component, e.g., an antibody or small molecule withaffinity for the super-enhancer component to form a complex between thecross-linked complex and the ligand;

optionally, identifying or sequencing chromosomal nucleic acid in thecomplex between the cross-linked complex and the ligand, therebyidentifying a super-enhancer, or a gene associated with asuper-enhancer.

In an embodiment the method comprises fragmenting the chromosomalnucleic acid, e.g., after the step of forming a cross-linked complex, orafter forming the complex between the cross-linked complex and theligand.

In embodiments the method comprises identifying a gene associated withthe super-enhancer.

In embodiments the method comprises classifying an enhancer as having afirst or second level of occupancy, wherein said first level is higher,e.g., 2, 5, 10, or 100 times higher than the second level.

In some aspects, the invention relates to a method of identifying asuper-enhancer, or a gene associated with a super-enhancer, comprising:

identifying sites on a segment of chromosome that are hypersensitive toreaction with an agent, e.g., a nuclease, e.g., a DNase, e.g., DNase 1;

identifying or sequencing chromosomal nucleic acid adjacent the sites;thereby identifying a super-enhancer, or a gene associated with asuper-enhancer.

In an embodiment the method comprises fragmenting the chromosomalnucleic acid, e.g., after the step of forming a cross-linked complex, orafter forming the complex between the cross-linked complex and theligand.

In embodiments the method comprises identifying a gene associated withthe super-enhancer.

In an embodiment, the method comprises confirming, e.g., by sequencing,that a candidate super-enhancer site comprises a plurality of enhancers.

In embodiments the method comprises classifying an enhancer as having afirst or second level of occupancy, wherein said first level is higher,e.g., 2, 5, 10, or 100 times higher than the second level.

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane,D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1988; Freshney, R.I., “Culture of Animal Cells, AManual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J.,2005. Non-limiting information regarding therapeutic agents and humandiseases is found in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic andClinical Pharmacology, McGraw-Hill/Appleton & Lange; 10^(th) ed. (2006)or 11th edition (July 2009). Non-limiting information regarding genesand genetic disorders is found in McKusick, V. A.: Mendelian Inheritancein Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: JohnsHopkins University Press, 1998 (12th edition) or the more recent onlinedatabase: Online Mendelian Inheritance in Man, OMIM™. McKusick-NathansInstitute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)and National Center for Biotechnology Information, National Library ofMedicine (Bethesda, Md.), as of May 1, 2010, World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), athttp://omia.angis.org.au/contact.shtml. All patents, patentapplications, and other publications (e.g., scientific articles, books,websites, and databases) mentioned herein are incorporated by referencein their entirety. In case of a conflict between the specification andany of the incorporated references, the specification (including anyamendments thereof, which may be based on an incorporated reference),shall control. Standard art-accepted meanings of terms are used hereinunless indicated otherwise. Standard abbreviations for various terms areused herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A, 1B, 1C and 1D demonstrate that Oct4/Sox2/Nanog defineenhancers in ES cells. FIG. 1A depicts an example enhancer upstream ofthe Hkt2 gene. FIG. 1B depicts an example of super-enhancer upstream ofthe Klf4 gene. FIG. 1C is a scatter plot showing Mediator occupancyacross the ˜6,400 ESC enhancers. FIG. 1D illustrates metagenes of Med1at typical and super-enhancers in ESCs.

FIGS. 2A, 2B, 2C and 2D demonstrate that Super-enhancers drive keypluripotency genes. FIG. 2A demonstrates that super-enhancers drivehighly expressed genes. FIG. 2B depicts an example of typicalenhancer-associated gene (with RNA-seq). FIG. 2C depicts an example of asuper-enhancer-associated gene (with RNA-seq). FIG. 2D illustrates thatsuper-enhancers associate with the ESC master regulators Oct4, Sox2 andNanog.

FIGS. 3A, 3B and 3C demonstrate that super-enhancers confer highenhancer activity. FIG. 3A shows OSN and Mediator gene tracks atenhancers near Klf4 (super-enhancer associated gene), and Egln3 (typicalenhancer-associated gene), and corresponding DNA binding motifs. FIG. 3Bdemonstrates that super-enhancers have high enhancer activity in vitro.3000 bp genomic fragments were cloned into a luciferase reporterplasmid. Luciferase activity was measured 24 hours post transfection,and was normalized to a co-transfected control plasmid. FIG. 3C depictsthe creation of artificial super-enhancers by clustering. Singleenhancers were genetically oligomerized and cloned into luciferasereporters. Luciferase activity was measured 24 hours post transfection,and was normalized to a co-transfected control plasmid.

FIGS. 4A and 4B demonstrate rapid loss of ESC super-enhancers andestablishment of new super-enhancers during ESC differentiation. FIG. 4Ais a cartoon diagram depicting treatment of ZHBTc4 ESCs with doxycyclineleading to loss of Oct4 proteins, loss of ESC state, and formation ofearly trophectoderm cells. FIG. 4B illustrates that Mediator is rapidlylost at key ESC super-enhancers compared to median enhancers. Bar graphsof mean normalized Med1 density before and during ESC differentiation atselected ESC super-enhancers and median enhancers. The associated geneswere identified based on their proximity to the enhancers. Asterisksdenote enhancers displaying at least two-fold reduction in Mediator.

FIGS. 5A, 5B, 5C, 5D and 5E demonstrate that super-enhancers are ageneral feature of mammalian cells and are cell-type specific. FIG. 5Ademonstrates that Pro-μ enhancers are associated with the gene Rag1.ChIP-Seq binding profiles (normalized reads/million) for the pro-μtranscription factor (Pu.1), and the Mediator coactivator (Med1) at theRag1 locus in pro-μ cells, with the y-axis floor set to 1. Gene model,and previously described enhancer regions are depicted below the bindingprofiles. FIG. 5B demonstrates that pro-μ super-enhancers are associatedwith the key pro-μ gene Inpp5d. ChIP-Seq binding profiles (normalizedreads/million) for the pro-μ transcription factor (Pu.1), and theMediator coactivator (Med1) at the Inpp5d locus in pro-μ cells, with they-axis floor set to 1. Gene model, and previously described enhancerregions are depicted below the binding profiles. FIG. 5C is a scatterplot of Mediator occupancy across the ˜13000 pro-μ enhancers. FIG. 5Ddemonstrates that master transcription factors (Oct4 for ESCs; Pu.1 forpro-B cells) and Mediator occupy approximately super-enhancer regionsthat are specific for ESCs and pro-B cells. Density maps of the Mediatorcoactivator (Med1) in ESCs and pro-B cells. Color scale reflectsChIP-Seq signal in reads per million. FIG. 5E demonstrates thatsuper-enhancer associated genes display highly cell-type specificpatterns of expression. Venn diagram of ESC super-enhancer-associatedgenes and pro-B super-enhancer-associated genes.

FIGS. 6A, 6B, 6C and 6D demonstrate that Mediator and BRD4 co-occupypromoters of active genes in multiple myeloma. FIG. 6A depicts genetracks of BRD4, MED1, H3K27ac, and H3K4me3 binding at the XBP1 gene inMM.1S multiple myeloma. FIG. 6B is a meta-gene representation of globalBRD4, MED1, H3K27ac, and H3K4me3 occupancy at enhancers and promoters.The top 5,000 active enhancers are defined by MED1 occupancy, and TSSincludes all transcriptionally active promoters defined by H3K4me3 andPOL2. FIG. 6C demonstrates that Mediator and BRD4 occupancy correlatewith one another at both enhancers and transcription start sites.Scatter plots depicting MED1 and BRD4 aggregate signal +/−5 kb fromenhancers and promoters (as defined in 1B). FIG. 6D demonstrates thatBRD4 occupancy at genes correlates with RNAPII levels.

FIGS. 7A, 7B, 7C and 7D demonstrate that super-enhancers are associatedwith key multiple myeloma genes. FIG. 7A demonstrates that the sizes ofenhancers occupied by Mediator show an unusual distribution. FIG. 7Bdepicts occupancy of MED1, BRD4, and H3K27ac at super-enhancers comparedto normal enhancers. FIG. 7C demonstrates that super-enhancers areassociated with highly expressed, cell type specific genes. FIG. 7Ddemonstrates that the IgH-MYC locus and IRF4 contain a largesuper-enhancers occupied by high levels of BRD4 and MED1.

FIGS. 8A, 8B, 8C, 8D, 8E and 8F demonstrate that BRD4 occupancy atsuper-enhancers is highly sensitive to bromodomain inhibition. FIG. 8Adepicts measuring the effects of various concentrations of JQ1 ongenome-wide on BRD4 occupancy. Schematic depicting the experimentalprocedure. FIG. 8B demonstrates that short-term JQ1 treatment (6 hours)has little effect on MM.1S cell viability. JQ1 sensitivity of MM.1Scells by measurement of ATP levels (CellTiterGlo) after 6 hours oftreatment. FIG. 8C illustrates that c-Myc protein levels aresignificantly depleted by JQ1 treatment. Western blot of relative c-MYClevels after 6 hours of JQ1 or DMSO treatment. FIG. 8D demonstrates thatJQ1 does not alter BRD4 levels or ChIP-efficency. Western blot ofrelative BRD4 levels after 6 hours of JQ1 or DMSO treatment.ChIP-Western blot of the relative levels of immunoprecipitated BRD4after 6 hours of JQ1 or DMSO treatment. FIG. 8E demonstrates thatsuper-enhancers show a greater loss of BRD4 occupancy when compared toregions with average or low amounts of BRD4. FIG. 8F demonstrates thatthe IgH enhancer shows significantly greater loss of BRD4 than regionswith lower BRD4 occupancy. Gene tracks of BRD4 at the IGH super enhancerand the average, CD28 enhancer after 6 hours of DMSO or JQ1 treatment.

FIGS. 9A, 9B and 9C demonstrate that the loss of P-TEFb accompanies BRD4inhibition. FIG. 9A demonstrates that P-TEFb generally occupiesenhancers bound by Mediator and BRD4 in MM1.S cells. FIG. 9Bdemonstrates that the loss of BRD4 following JQ1 treatment isaccompanied by loss of P-TEFb at enhancers. FIG. 9C demonstrates thatP-TEFb is disproportionally lost at super-enhancers.

FIGS. 10A, 10B and 10C demonstrate that JQ1 causes disproportionate lossof transcription at super-enhancer genes. FIG. 10A demonstrates that JQ1leads to a global defect in transcription elongation. FIG. 10Bdemonstrates that genes associated with super-enhancers show a dramaticdefect in elongation. Gene tracks of RNA PolII occupancy at the MYC geneafter 6 hour treatment with JQ1. FIG. 10C demonstrates that genesassociated with super enhancers show a larger increase in travellingratio in response to JQ1 compared to genes associated with normalenhancers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates in some aspects to super-enhancers andrelated compositions, methods, and agents that are useful for modulatingexpression of cell type-specific genes that are required for maintenanceof cell identity (e.g., embryonic stem cell identity) or maintenance ofa disease state (e.g., cancer). In some aspects, the present inventionrelates to methods of identifying super-enhancers,super-enhancer-associated genes, and disease-related genes in cells,tissues, organs and individuals, and kits comprising reagents forperforming those methods.

During the course of work described herein, more than 200 genomicregions that contained tightly spaced clusters of enhancers spanningextraordinarily large domains were discovered. These “super-enhancers”are occupied by more transcriptional coactivator (e.g., Mediator) thanthe average or median enhancers, exhibit greater activity than averageenhancers, and are sufficient to drive high expression of key, celltype-specific genes required to maintain cell identity or disease state.

Accordingly, in some aspects, the present invention relates to anisolated super-enhancer, or functional fragment and/or variant thereof,comprising a genomic region of DNA that contains at least two enhancers,wherein the genomic region is occupied when present within a cell bymore super-enhancer component, e.g., transcriptional coactivator thanthe average single enhancer within the cell. As used herein, “enhancer”refers to a short region of DNA to which proteins (e.g., transcriptionfactors) bind to enhance transcription of a gene. As used herein,“transcriptional coactivator” refers to a protein or complex of proteinsthat interacts with transcription factors to stimulate transcription ofa gene. In some embodiments, the transcriptional coactivator isMediator. In some embodiments, the transcriptional coactivator is Med1(Gene ID: 5469). In some embodiments, the transcriptional coactivator isa Mediator component. As used herein, “Mediator component” comprises orconsists of a polypeptide whose amino acid sequence is identical to theamino acid sequence of a naturally occurring Mediator complexpolypeptide. The naturally occurring Mediator complex polypeptide canbe, e.g., any of the approximately 30 polypeptides found in a Mediatorcomplex that occurs in a cell or is purified from a cell (see, e.g.,Conaway et al., 2005; Kornberg, 2005; Malik and Roeder, 2005). In someembodiments a naturally occurring Mediator component is any of Med1-Med31 or any naturally occurring Mediator polypeptide known in the art. Forexample, a naturally occurring Mediator complex polypeptide can be Med6,Med7, Med10, Med12, Med14, Med15, Med17, Med21, Med24, Med27, Med28 orMed30. In some embodiments a Mediator polypeptide is a subunit found ina Med11, Med17, Med20, Med22, Med 8, Med 18, Med 19, Med 6, Med 30, Med21, Med 4, Med 7, Med 31, Med 10, Med 1, Med 27, Med 26, Med14, Med15complex. In some embodiments a Mediator polypeptide is a subunit foundin a Med12/Med13/CDK8/cyclin complex. Mediator is described in furtherdetail in PCT International Application No. WO 2011/100374, theteachings of which are incorporated herein by reference in theirentirety. In some embodiments, Mediator occupation of an enhancer, e.g.,a superenhancer, may be detected by detecting one or more Mediatorcomponents. It is to be understood that a Mediator inhibitor may inhibitone or more Mediator components or inhibit interaction(s) between themor inhibit interaction with a transcription factor.

In some embodiments a “naturally occurring polypeptide” is a polypeptidethat naturally occurs in a eukaryote, e.g., a vertebrate, e.g., amammal. In some embodiments the mammal is a human. In some embodimentsthe vertebrate is a non-human vertebrate, e.g., a non-human mammal,e.g., rodent, e.g., a mouse, rat, or rabbit. In some embodiments thevertebrate is a fish, e.g., a zebrafish. In some embodiments theeukaryote is a fungus, e.g., a yeast. In some embodiments the eukaryoteis an invertebrate, e.g., an insect, e.g., a Drosophila, or a nematode,e.g., C. elegans. Any eukaryotic species is encompassed in variousembodiments of the invention. Similarly a cell or subject can be of anyeukaryotic species in various embodiments of the invention. In someembodiments, the sequence of the naturally occurring polypeptide is thesequence most commonly found in the members of a particular species ofinterest. One of skill in the art can readily obtain sequences ofnaturally occurring polypeptides, e.g., from publicly availabledatabases such as those available at the National Center forBiotechnology Information (NCBI) website (e.g., GenBank, OMIM, Gene).

In some embodiments, the transcriptional coactivator is a component ofMediator. In some embodiments, the Mediator component comprises a Med1or a Med12 polypeptide. In some embodiments, the at least one Mediatorcomponent comprises Med6, Med7, Med10, Med12, Med14, Med15, Med17,Med21, Med24, Med27, Med28 and Med30 polypeptides.

In some embodiments, the genomic region of the super-enhancer isoccupied when present within a cell by more chromatin regulator than theaverage single enhancer within the cell. As used herein, “chromatinregulator” refers to a protein or complex of proteins that is involvedin regulating gene expression by interacting with transcription factors,transcriptional coactivators, and/or acetylated histone residues in away that modulates expression of a super-enhancer-associated gene. Insome instances, the chromatin regulator possesses histoneacetyltransferase (HAT) activity. HATs are responsible for acetylatinglysine residues on histone tails of nucleosomes, thereby relaxing thechromatin and increasing access to DNA. In some embodiments, thechromatin regulator is a BET bromodomain protein. In some embodiments,the BET bromodomain protein is BRD4 (Gene ID: 23476).

Generally, super-enhancers formed by the at least two enhancers in thegenomic region of DNA are of greater length than the average singleenhancer. In some embodiments, the length of the genomic region thatforms the super-enhancer is at least an order of magnitude greater thanthe average single enhancer. In some embodiments, the genomic regionspans between about 4 kilobases and about 40 kilobases in length. Itshould be appreciated, however, that super-enhancers may comprisegenomic regions less than 4 kilobases or greater than 40 kilobases inlength, as long as the genomic region contains clusters of enhancersthat can be occupied when present within a cell by extremely high levelsof a transcriptional coactivator (e.g., Mediator).

Table 1 (relating to nucleotide sequences of super-enhancers foundwithin embryonic stem cells) and Table 2 (relating to nucleotidesequences of super-enhancers found within multiple myeloma cells); Table3 (relating to nucleotide sequences of super-enhancers found inglioblastoma cells); and Table 4 (relating to nucleotide sequences ofsuper-enhancers found in SCLC cells) disclose information that can berelied upon by one of skill in the art to obtain the specific nucleotidesequences for exemplary super-enhancers of the invention. For example,using the chromosomal number, and start and stop positions, as well asthe sense orientation (e.g. +) of the sequence provided in Tables 1 and2, one of skill in the art would be able to utilize a publicly availabledatabase (e.g., USCS Genome Browser, available at genome.ucsc.edu/) toobtain the nucleotide sequences of the specified super-enhancers. Forthe embryonic stem cell super-enhancer nucleotide sequences specified inTable 1, the mm9 genome build was used. This corresponds to NCBI build37. For the multiple myeloma cell super-enhancer nucleotide sequencesspecified in Table 2, the hg 18 genome build was used. This correspondsto NCBI build 36. Tables 3 and 4 are also based on the hg 18 genomebuild. In some embodiments, the invention comprises a super-enhancer, orfunctional fragment and/or variant thereof, having a nucleotide sequencespecified in Tables 1, 2, 3 or 4.

The at least two enhancers which form the super-enhancers, or functionalfragment and/or variant thereof, are clustered together.

It should be appreciated that the each of the at least two enhancers canbe the same type of enhancer or the at least two enhancers can bedifferent types of enhancers. Each enhancer of the at least twoenhancers comprises a binding site for a cognate transcription factorthat interacts with the transcriptional coactivator to stimulatetranscription of the gene associated with the super-enhancer. In someembodiments, the cognate transcription factor comprises an embryonicstem cell master transcription factor. Examples of suitable embryonicstem cell master transcription factors include, but are not limited toOct4, Sox2, Nanog, Esrrb, Utf1, Klf4, mir-290-295 gene cluster, Tbx3,Sgk1, and combinations thereof. In some embodiments, the cognatetranscription factor comprises an oncogenic transcription factor.Examples of suitable oncogenic transcription factors include, but arenot limited to c-Myc, IRF4, p53, AP-1, Bcr-Abl, c-Fos, c-Jun andcombinations thereof. In some embodiments, the cognate transcriptionfactor comprises a muscle cell transcription factor, for example,transcription factor MyoD. In some embodiments, the cognatetranscription factor comprises a B cell transcription factor, forexample Pu.1.

As noted above, the genomic region of the super-enhancers are occupiedwhen present within a cell by more transcriptional coactivator (e.g.,Mediator) and/or more chromatin regulator (e.g., BRD4) than the averagesingle enhancer within the cell. In some embodiments, the genomic regionof a super-enhancers is occupied when present within the cell by anorder of magnitude more transcriptional coactivator or chromatinregulator than the average single enhancer in the cell. As used herein,“order of magnitude” refers to the relative fold difference in a featureor classification of one object as compared to a feature orclassification of another object (e.g., a level or an amount oftranscriptional coactivator occupying a super-enhancer associated with agene as compared to the level or the amount of transcriptionalcoactivator occupying the average or median enhancer associated with thegene). In some embodiments, the order of magnitude is at least 1-fold,2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-foldor more. In some embodiments, the order of magnitude is at least 2-fold(i.e., there is a 2-fold greater amount of transcriptional coactivatoroccupying the super-enhancer associated with a gene than the amount oftranscriptional coactivator occupying the average enhancer in the gene).In some embodiments, the order of magnitude is at least 10-fold. In someembodiments, the order of magnitude is at least 15-fold. In someembodiments, the order of magnitude is at least 16-fold.

Work described herein suggests that super-enhancers are a common featureof mammalian cells. Accordingly, the present invention contemplates thatsuper-enhancers can be isolated from any mammalian cell type. Suchisolation can be achieved by routine methods well known to those skilledin the art.

In some embodiments, super-enhancers of the present invention can beused drive high levels of expression of cell type specific genes. A celltype specific gene is typically expressed selectively in one or a smallnumber of cells types relative to expression in many or most other celltypes. One of skill in the art will be aware of numerous genes that areconsidered cell type specific. A cell type specific gene need not beexpressed only in a single cell type but may be expressed in one orseveral, e.g., up to about 5, or about 10 different cell types out ofthe approximately 200 commonly recognized (e.g., in standard histologytextbooks) and/or most abundant cell types in an adult vertebrate, e.g.,mammal, e.g., human. In some embodiments, a cell type specific gene isone whose expression level can be used to distinguish a cell of one ofthe following types from cells of the other cell types: adipocyte (e.g.,white fat cell or brown fat cell), cardiac myocyte, chondrocyte,endothelial cell, exocrine gland cell, fibroblast, glial cell,hepatocyte, keratinocyte, macrophage, monocyte, melanocyte, neuron,neutrophil, osteoblast, osteoclast, pancreatic islet cell (e.g., a betacell), skeletal myocyte, smooth muscle cell, B cell, plasma cell, T cell(e.g., regulatory, cytotoxic, helper), or dendritic cell. In someembodiments a cell type specific gene is lineage specific, e.g., it isspecific to a particular lineage (e.g., hematopoietic, neural, muscle,etc.) In some embodiments, a cell-type specific gene is a gene that ismore highly expressed in a given cell type than in most (e.g., at least80%, at least 90%) or all other cell types. Thus specificity may relateto level of expression, e.g., a gene that is widely expressed at lowlevels but is highly expressed in certain cell types could be consideredcell type specific to those cell types in which it is highly expressed.It will be understood that expression can be normalized based on totalmRNA expression (optionally including miRNA transcripts, long non-codingRNA transcripts, and/or other RNA transcripts) and/or based onexpression of a housekeeping gene in a cell. In some embodiments, a geneis considered cell type specific for a particular cell type if it isexpressed at levels at least 2, 5, or at least 10-fold greater in thatcell than it is, on average, in at least 25%, at least 50%, at least75%, at least 90% or more of the cell types of an adult of that species,or in a representative set of cell types. One of skill in the art willbe aware of databases containing expression data for various cell types,which may be used to select cell type specific genes. In someembodiments a cell type specific gene is a transcription factor.

In some aspects, the present invention relates to a compositioncomprising a super-enhancer of the present invention or a functionalvariant thereof. Such compositions may be useful for stimulating theexpression of a gene or genes in a specific cell type, for example, tostimulate the expression of embryonic stem cell master transcriptionfactors to maintain the cell in an embryonic stem cell-like state. Insome instances, such compositions may be useful for stimulating theexpression of a gene or genes in a specific cell type to change theidentity of a specific cell-type, for example, by introducing asuper-enhancer associated with a differentiated state to change theidentity of an embryonic stem cell to a more differentiated state. Insome embodiments, the super-enhancer can be used to stimulate expressionof a target gene that is to be transfected into a cell for in vitroexpression of that target gene. In some embodiments, the super-enhancercan be used to simulate a disease like state. By way of example, and notof limitation, an super-enhancer can be constructed using enhancers ofan oncogene and transfection of the oncogene with the artificialenhancer can be useful to simulate the disease associated with theoncogene. Another exemplary use of a super-enhancer of the presentinvention is to identify genes that are prone to lead to disease uponaberrant expression. Such super-enhancers may be used in cells, tissues,organs, and whole organisms to artificially increase the expression ofcertain genes and examine the biological effects that the increasedexpression of the gene has on the cell, the tissue, organ, or animal.

It should be appreciated that any enhancer associated with the targetgene can be cloned and used to form the super-enhancers. In someembodiments, the super-enhancer is engineered to mimic a super-enhanceridentified in vivo, such as a super-enhancer that is responsible formaintaining embryonic stem cell identity, i.e., a super-enhancercomprising a plurality of Oct4, Sox2, and Nanog binding motifsoligomerized to form a concatemer.

In some aspects, the present invention relates to a nucleic acidconstruct comprising a super-enhancer, or functional fragment thereof,of the present invention. Methods of forming nucleic acid constructs areknown to those skilled in the art. It should be understood that thenucleic acid constructs of the present invention are artificial orengineered constructs not to be confused with native genomic sequences.Such nucleic acid constructs can be used, for example, to increase theexpression of a gene or genes associated with or regulated by thesuper-enhancer in the nucleic acid construct. In some instances, anucleic acid construct comprising the super-enhancer can be introducedinto a target cell and the super-enhancer can interact with endogenouscellular components to drive expression of an endogenous gene within thecell. In some embodiments, the nucleic acid construct includes anucleotide sequence encoding a target gene operatively linked to thesuper-enhancer. In such instances, the nucleic acid can be transfectedinto a cell and interact with endogenous cellular components to driveexpression of the exogenous target gene associated with thesuper-enhancer. In other embodiments, the nucleic acid construct caninclude a nucleic acid sequence encoding a transcriptional coactivatoror chromatin regulator that can be expressed within the cell to producetranscriptional coactivator or chromatin regulator that can occupy thegenomic region of the super-enhancer and increase expression of the geneassociated with the super-enhancer in the cell. In some embodiments, thenucleic acid can include a reporter.

In some embodiments a reporter comprises a nucleic acid sequence thatencodes a detectable marker, e.g., a fluorescent protein such as greenfluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyanfluorescent proteins and fluorescent variants such as enhanced GFP(eGFP), mFruits such as mCherry, mTomato, mStrawberry; R-Phycoerythrin,etc. Enzymes useful as reporters include, e.g., enzymes that act on asubstrate to produce a colored, fluorescent, or luminescent substance.Examples include luciferases, beta-galactosidase, horseradishperoxidase, and alkaline phosphatase. In some embodiments, alteration(e.g., reduction) in the level of a reporter may be used to identify acompound that modulates (e.g., inhibits) activity of a super-enhancer.

In some aspects, the present invention relates to a kit for increasingthe expression of a gene, the kit including one or more or all of: (a) apopulation of cells; (b) reagents suitable for culturing said populationof cells; (c) a nucleic acid construct comprising a super-enhancerenhancer or functional fragment and/or variant thereof, and a geneassociated with the super-enhancer enhancer or functional fragmentand/or variant thereof, that is capable of being expressed within saidpopulation of cells; and optionally (d) transcriptional coactivator orchromatin regulator e.g., excess levels of transcriptional coactivatoror chromatin regulator that, e.g., can be introduced into saidpopulation of cells such that an order of magnitude more transcriptionalcoactivator or chromatin regulator occupies enhancers clustered withinthe super-enhancer and increases the expression of the gene within thecells.

In some aspects, the present invention relates to a cell, or cell-freesystem, into which a super-enhancer is introduced, for example bytransfection of a nucleic acid construct comprising the super-enhancer,wherein upon introduction of super-enhancer into the cell, or cell-freesystem, endogenous transcriptional coactivators and chromatin regulatorswithin the cell co-occupy the enhancer clusters of the super-enhancerand the active transcription start sites of the target gene to stimulateexpression of the target gene within the cell. It should be appreciatedthat the super enhancer, or functional fragment and/or variant thereof,may be associated with and regulate an endogenous gene within thetransfected cell. In such instances, the gene regulated by thesuper-enhancer, or functional fragment and/or variant thereof, need notbe introduced into the cell with the super-enhancer, for example anucleic acid construct need not include a target gene for expressionwithin the transfected cell. In other instances, such as when anexogenous gene is desired to be introduced within the transfected cell,or cell-free system, the exogenous gene can be introduced into the cellwith the super-enhancer, or functional fragment and/or variant thereof,or functional fragment and/or variant thereof. It should be appreciatedthat the exogenous gene and the super-enhancer or functional fragmentand/or variant thereof, can be introduced into the cell by any methodand in any form (e.g., protein or nucleic acid). The exogenous gene andthe super-enhancer, or functional fragment and/or variant thereof, canbe introduced into the cell, or cell-free system, together orseparately, for example a nucleic acid construct comprising the superenhancer, or functional fragment and/or variant thereof, may be furtherengineered to include an exogenous gene operatively linked to thesuper-enhancer, or functional fragment and/or variant thereof, and whichis also capable of being expressed within the transfected cell, orcell-free system. In some embodiments, exogenous transcriptionalcoactivators and/or chromatin regulators can be introduced into thetransfected cell, or cell-free system to ensure that the enhancerclusters of the super-enhancer and the active transcription start sitesare co-occupied within the transfected cell, or cell-free system by moretranscriptional coactivator and/or the chromatin regulator and therebydrive high levels of expression of either an exogenous or endogenousgene in the transfected cell, or cell-free system.

The super-enhancer and/or a nucleic acid construct comprising thesuper-enhancer, or functional fragment and/or variant thereof, can betransfected into any cell suitable for expressing the gene associatedwith the super-enhancer. In some embodiments, the cell is a mammaliancell. In some embodiments, the cell is a human cell. In someembodiments, the cell is an embryonic stem cell or embryonic stemcell-like cell. In some embodiments, the cell is a muscle cell. In someembodiments, the muscle cell is a myotube. In some embodiments, the cellis a B cell. In some embodiments, the B cell is a Pro-B cell.

In some aspects, the present invention relates to a functional variantof a super-enhancer. A variant may be shorter or longer than theoriginal super-enhancer. The term “variant” encompasses “fragments” or“functional fragments” of super-enhancers, or functional sequencevariants, of super-enhancers. A “fragment” is a continuous portion of apolypeptide or polynucleotide that is shorter than the originalpolypeptide or polynucleotide. In some embodiments a variant comprisesor consists of a fragment. In some embodiments a fragment or variant isat least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or more as long as the original polypeptide or polynucleotide. Afragment may be an N-terminal, C-terminal, or internal fragment. Afunctional fragment of a super-enhancer can have one or more of thefollowing properties:

a) when associated with a gene, e.g., a gene with which it is normallyassociated, it provides at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%of the level of expression as is seen with the intact super-enhancer;

b) when associated with a gene, e.g., a gene with which it is normallyassociated, it provides at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%of the level of binding of an super-enhancer component;

c) when associated with a gene, e.g., a gene with which it is normallyassociated, it provides at least 10, 20, 30, 40, 50, 60, 70, 80, or 90%of the level of binding of a mediator protein, e.g., Med1;

d) it comprises at least 10, 20, 30, 40, 5, 60, 70, 80 or 90% of theenhancers of the super-enhancer of which it is a functional fragment; or

e) it is at least 10, 20, 30, 40, 5, 60, 70, 80 or 90% as long as thesuper-enhancer of which it is a functional fragment.

The term variant also encompasses “sequence variants,” e.g., “functionalsequence variants,” of a super enhancer or fragment or functionalfragment of a super-enhancer. A functional sequence variant of asuper-enhancer can have one or more of the following properties:

a) it comprises sufficient nucleotide sequence homology or identity witha reference super-enhancer, e.g., the super-enhancer from which it isderived, that when associated with a gene, e.g., a gene with which thereference super-enhancer is normally associated, it provides at least10, 20, 30, 40, 50, 60, 70, 80, or 90% of the level of expression as isseen with the reference super-enhancer;

b) when associated with a gene, e.g., a gene with which the referencesuper-enhancer, e.g., the super-enhancer from which it is derived, isnormally associated, it provides at least 10, 20, 30, 40, 50, 60, 70,80, or 90% of the level of binding of an super-enhancer component as isseen with the reference super-enhancer;

c) when associated with a gene, e.g., a gene with which the referencesuper-enhancer, e.g., the super-enhancer from which it is derived, isnormally associated, it provides at least 10, 20, 30, 40, 50, 60, 70,80, or 90% of the level of binding of a mediator protein, e.g., Med1 asis seen with the reference super-enhancer;

d) it comprises at least 10, 20, 30, 40, 5, 60, 70, 80 or 90% of thenumber of functional enhancers as is seen with the referencesuper-enhancer, e.g., the super-enhancer from which it is derived;

e) it comprises at least 40, 50, 60, 70, 80, 90, 95, 97, or 99% sequencehomology or identity with a reference super-enhancer, e.g., thesuper-enhancer from which it is derived;

f) it comprises at least 40, 50, 60, 70, 80, 90, 95, 97, or 99% sequencehomology or identity, across its encompassed enhancer elements, with areference super-enhancer, e.g., the super-enhancer from which it isderived; or

f) it comprises a first level or sequence or homology or identity acrossits encompassed enhancer elements and/or associated protein encodingelement, and a second level of homology across untranslated and/oruntranscribed regions between its encompassed enhancers, with areference super-enhancer, e.g., the super-enhancer from which it isderived, wherein the first and second levels are independently selectedfrom at least 40, 50, 60, 70, 80, 90, 95, 97, or 99% sequence homologyor identity, and, e.g., the first level is higher than the second level,e.g., the first level is at least 80, 90, 95, 97, or 99% and the secondlevel is at least 40, 50, or 60%.

In some embodiments a variant polypeptide comprises or consists of atleast one domain of an original polypeptide. In some embodiments avariant polynucleotide hybridizes to an original polynucleotide understringent conditions, e.g., high stringency conditions, for sequences ofthe length of the original polypeptide. In some embodiments a variantpolypeptide or polynucleotide comprises or consists of a polypeptide orpolynucleotide that is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, or more identical in sequence to the original polypeptide orpolynucleotide over at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, 96%, 97%, 98%, 99%, or 100% of the original polypeptide orpolynucleotide. In some embodiments a variant polypeptide comprises orconsists of a polypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or more identical in sequence to the originalpolypeptide over at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100% of the original polypeptide, with theproviso that, for purposes of computing percent identity, a conservativeamino acid substitution is considered identical to the amino acid itreplaces. In some embodiments a variant polypeptide comprises orconsists of a polypeptide that is at least 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or more identical to the original polypeptide overat least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or 100% of the original polypeptide, with the proviso that any oneor more amino acid substitutions (up to the total number of suchsubstitutions) may be restricted to conservative substitutions. In someembodiments a percent identity is measured over at least 100; 200; 300;400; 500; 600; 700; 800; 900; 1,000; 1,200; 1,500; 2,000; 2,500; 3,000;3,500; 4,000; 4,500; or 5,000 amino acids. In some embodiments thesequence of a variant polypeptide comprises or consists of a sequencethat has N amino acid differences with respect to an original sequence,wherein N is any integer between 1 and 10 or between 1 and 20 or anyinteger up to 1%, 2%, 5%, or 10% of the number of amino acids in theoriginal polypeptide, where an “amino acid difference” refers to asubstitution, insertion, or deletion of an amino acid. In someembodiments a difference is a conservative substitution. Conservativesubstitutions may be made, e.g., on the basis of similarity in sidechain size, polarity, charge, solubility, hydrophobicity, hydrophilicityand/or the amphipathic nature of the residues involved. In someembodiments, conservative substitutions may be made according to TableA, wherein amino acids in the same block in the second column and in thesame line in the third column may be substituted for one another otherin a conservative substitution. Certain conservative substitutions aresubstituting an amino acid in one row of the third column correspondingto a block in the second column with an amino acid from another row ofthe third column within the same block in the second column.

TABLE A Aliphatic Non-polar G A P I L V Polar - uncharged C S T M N QPolar - charged D E K R Aromatic H F W Y

In some aspects, the present invention relates to a method of increasingthe level of expression of a target gene in a cell, the method includingtransfecting a cell under conditions suitable for expression of thetarget gene with a nucleic acid expression construct comprising anucleic acid sequence encoding the target gene operatively linked to asuper-enhancer, wherein upon transfection of the cell endogenoustranscriptional coactivators and chromatin regulators within the cellco-occupy enhancers clustered within the super-enhancer and activetranscription start sites of the target gene to increase the level ofexpression of the target gene within the cell. Those skilled in the artwill appreciate that the step of transfecting can be achieved in avariety of ways according to well-known and routine methods, forexample, by using a transfection reagent, such as a plasmid or a lipidbased transfection reagent. In some instances, it may be desirable tointroduce into the cell exogenous transcriptional coactivators andchromatin regulators to ensure that enhancers clustered within thesuper-enhancer and the activate transcription start sites of the targetgene are co-occupied by an order of magnitude more of thetranscriptional coactivators and chromatin regulators than the averageenhancer of the target gene. The exogenous transcriptional coactivatorsand chromatin regulators can be introduced into the target cell in theform of nucleic acids that can be transfected into the cell forexpression within the cell or in the form of proteins, for example, bymicroinjecting the proteins into the cell. Other ways of introducingnucleic acids and proteins into a cell are apparent to those skilled inthe art. Upon transfection of the cell with the nucleic acid constructcontaining the super enhancer, or functional fragment and/or variantthereof, it is expected that the level of expression of the target genewill increase significantly, for example, the level of expression of thetarget gene is increased 2-fold, 3-fold, 4-fold, 5-fold, 6-fold or morewithin the cell.

In some aspects, the present invention relates to a kit for increasingthe expression of a target gene in a cell, comprising one or more or allof: (a) a super-enhancer operatively linked to a target gene; (b) apopulation of cells suitable for expression of said target gene; and (c)a reagent for introducing the super-enhancer and the target gene intosaid population of cells. In some embodiments, the reagent comprises atransfection reagent, e.g., a plasmid.

In some aspects, the present invention relates to a kit for increasingthe expression of a target gene in a cell, comprising one or more or allof: (a) a nucleic acid construct comprising an artificial super-enhanceroperatively linked to the target gene; (b) a population of cellssuitable for expression of said target gene; and (c) a reagent fortransfecting said population of cells with said nucleic acid construct.

In some aspects, the invention relates to a nucleic acid vectorcomprising a super-enhancer, or functional fragment and/or variantthereof, and a site, e.g., a restriction enzyme site, disposed such thatinsertion of a structural gene at the site places the structural geneunder the control of the super-enhancer, or functional fragment and/orvariant thereof. In embodiments the vector further comprise one or moreof a first selectable marker, a second selectable marker, and an originof replication.

In some aspects, the invention relates to a nucleic acid vectorcomprising a super-enhancer or functional fragment and/or variantthereof, functionally linked to a heterologous reporter gene, e.g., afluorescent protein e.g., GFP, or an enzyme, e.g., horse radishperoxidase. In embodiments the vector further comprise one or more of afirst selectable marker, a second selectable marker, and an origin ofreplication.

In some aspects, the invention relates to a kit comprising one or bothof:

a first nucleic acid comprising a reference super-enhancer or functionalfragment and/or variant thereof, optionally, coupled to a reporter gene;and a second nuclide acid comprising a site for insertion of an SE, orfunctional fragment and/or variant thereof, optionally, coupled to areporter gene.

In some aspects, the invention relates to a kit comprising one or bothof:

a nucleic acid vector comprising a super-enhancer, or functionalfragment and/or variant thereof, functional linked to a heterologousreporter gene, e.g., a fluorescent protein e.g., GFP, or an enzyme,e.g., horse radish peroxidase. In embodiments the vector furthercomprise one or more of a first selectable marker, a second selectablemarker, and an origin of replication; and

a nucleic acid vector comprising an site, e.g., an restriction enzymesite, and a reporter gene, e.g., a fluorescent protein e.g., GFP, or anenzyme, e.g., horse radish peroxidase, wherein said site is disposedsuch that insertion into the site of a super-enhancer, or functionalfragment and/or variant thereof, will place the reporter gene under thecontrol of the super-enhancer, or functional fragment and/or variantthereof. In embodiments the vector further comprise one or more of afirst selectable marker, a second selectable marker, and an origin ofreplication.

In some aspects, the present invention relates to a method ofidentifying a super-enhancer in a cell, or cell-free system, comprising:(a) identifying a genomic region of a target gene within said cell, orcell-free system characterized by clusters of enhancers for bindingcognate transcription factors capable of interacting with Mediator tostimulate transcription of the target gene within said cell, orcell-free system; (b) measuring in the identified genomic region a levelof Mediator occupying said enhancers; and (c) identifying the genomicregion as a super-enhancer if the level of Mediator occupying theclusters of enhancers is an order of magnitude more than the level ofMediator occupying the average enhancer of the target gene.

In other aspects, the present invention relates to a method ofidentifying a super-enhancer associated with a target gene, comprising:(a) analyzing the target gene for a genomic region comprising clustersof enhancers occupied by an order of magnitude more Mediator than anaverage enhancer of the target gene; and (b) identifying the genomicregion as a super-enhancer associated with the target gene if saidclusters of enhancers are occupied by the order of magnitude moreMediator than the average enhancer of the target gene In someembodiments, the order of magnitude is at least 2-fold, 10-fold, atleast 15-fold, at least 16-fold, or more.

In some aspects, the present invention relates to a method ofidentifying a gene, e.g., a key gene or genes, that control a cell stateor identity, e.g., contributes to unwanted proliferation, e.g., whichcontributes to a cancerous cell state, comprising: (a) identifying asuper-enhancer, e.g., within an animal, cell, or cell-free system; and(b) identifying a gene or genes associated with the super-enhancer,e.g., a gene or genes within a range of proximity to the super-enhancer.

In an embodiment gene or genes that are within a certain proximity tothe super-enhancer are identified as a putative key gene or genes thatcontrol the cell state or identity.

In an embodiment the method is performed in a cell-free system.

In an embodiment the method is performed in a cell preparation, e.g., acultured cell preparation.

In an embodiment the method is performed in an animal model.

In an embodiment the method is first performed in a cell-free system,and repeated in a cell preparation, e.g., a cultured cell preparation.

In an embodiment the method is first performed in a cell-free system, ora cell preparation, e.g., a cultured cell preparation, and repeated inan animal.

In an embodiment the cell is a disease state cell, e.g., a cancer cell.

In an embodiment the cell-free system is derived from a disease statecell, e.g., a cancer cell.

In an embodiment, the identified gene is tested as a target for therapy,e.g., by administering an antagonist or inhibitor, e.g., an siRNA, ofthe product of the gene, to a cell or animal.

The range of proximity to the super-enhancer can extend as far as about10 megabases (mb) upstream to one end of the super-enhancer to as far asabout 10 mb downstream to the other end of the super-enhancer, and anyrange therebetween, for example 9 mb upstream to 9 mb downstream, 8 mbupstream to 8 mb downstream, 7 mb upstream to 7 mb downstream, 6 mbupstream to 6 mb downstream, 5 mb upstream to 5 mb downstream, 4 mbupstream to 4 mb downstream, 3 mb upstream to 3 mb downstream, 2 mbupstream to 2 mb downstream to 1 mb upstream to 1 mb downstream, orbetween 0.5 mb upstream and 0.5 mb downstream, 0.1 mb upstream to 0.1 mbdownstream. It should be appreciated that the key genes could also, insome instances, overlap with the super-enhancer region. It is also to beunderstood that the range of proximity will increase or decreasedepending on the length or size of the super-enhancer region, forexample, if the super enhancer is 10 kb in length, then the upstreamrange of proximity extends as far as about 10 mb upstream to the mostupstream portion of the 10 kb super-enhancer. Similarly, the downstreamrange of proximity would extend as far as about 10 mb downstream fromthe most downstream portion of the 10 kb super-enhancer. In someembodiments, the method of identifying key genes that control the cellstate or identity involves measuring the expression of those genes inthe cell in the presence and absence of an agent that disrupts thefunction of the super-enhancer identified, as well as assaying the cellfor changes in its cell state or identity (e.g., from a moredifferentiated state to a less differentiated state, or from a healthystate to a diseased state). If the expression of a gene within the rangeof proximity is statistically significant when the super-enhancer isproperly functioning but its expression decreases or becomesunremarkable in the presence of the agent that disrupts thesuper-enhancer function, then it is likely that the particular gene is akey gene that controls the cell state or identity, especially if itsabsence of expression is correlated to a change in the state or identityof the cell.

The aforementioned methods of identifying super-enhancers within a celland identifying a super-enhancer associated with a target gene can beachieved by a variety of different methods, as would be understood by aperson skilled in the art. In some embodiments, the super-enhancer isidentified by performing chromatin immunoprecipitation high-throughputsequencing (ChIP-Seq). Example 1 below describes an example of aprotocol that can be used to carry out such methods in normal cells,such as embryonic stem cells, for example. Example 2 below describes anexample of a protocol that can be used to carry out such methods intumor cells, such as MM.1S cells, for example.

In certain aspects, the present invention relates to a method ofidentifying a disease related super-enhancer in a cell, tissue, or organof an individual suspected of having said disease, comprising: (a)identifying a super-enhancer in said cell, tissue, or organ; (b)identifying a gene associated with said super-enhancer; and (c) andcorrelating said super-enhancer to said disease.

In certain aspects, the present invention relates to a method ofcharacterizing a subject, e.g, a subject having or suspected of having adisorder, e.g., a proliferative disorder, e.g., cancer, comprising:

acquiring a subject tissue sample;

determining if a super-enhancer is associated with a gene, e.g., apreselected gene,

thereby characterizing said subject.

In an embodiment the method comprises determining the genes in thesample that are associated with a super-enhancer.

In an embodiment, the patient is selected, classifed, diagnosed,treated, or prognosed, responsive to the pattern of genes, e.g., apreselected pattern, associated with a super-enhancers, e.g., where aplurality of genes, e.g., a plurality of preselected genes, areassociated with super-enhancers.

In an embodiment, the determination comprises: crosslinking chromatinfrom the sample, and selecting, e.g., by immunoprecipitation, a targetprotein, e.g., an super-enhancer component.

In an embodiment the target protein is a Mediator protein.

In an embodiment the gene or preselected gene is an oncogene, a kinase,a gene that controls cell proliferation, e.g., a myc gene.

In an embodiment the gene or preselected gene is other than an oncogene,a kinase, a gene that controls cell proliferation, e.g., a myc gene.

In an embodiment the method comprises classifying the subject as havinga super-enhancer associated with a gene, e.g., a preselected gene.

In an embodiment the sample comprises cancer cells.

In an embodiment the method comprises characterizing a subject by:

acquiring a subject tissue sample;

determining a gene in the sample is associated with a super-enhancer,

thereby characterizing said subject.

In an embodiment, responsive to said determination, the method comprisesselecting and/or administering a therapy to said subject.

In an embodiment, responsive to said determination, the method comprisesselecting, classifying, diagnosing, or prognosing said subject.

In an embodiment, responsive to said determination, the method comprisesclassifying the subject for treatment with an agent that antagonizes orinhibits the product of the gene or preselected gene.

In an embodiment, responsive to said determination, the method comprisesadministering to the subject an agent that antagonizes or inhibits theproduct of the gene or preselected gene.

A reaction mixture comprising a patient sample comprising chromatin froma cancer cell and a probe capable of determining if a preselected geneis associated with an super-enhancer.

In certain aspects, the present invention relates to a method ofmodifying a cell state or identity, comprising introducing into the cella super-enhancer that is required to stabilize the cell state oridentity. It is to be understood that the super-enhancers of the presentinvention are capable of modifying the cell state or identity of anycell in which it has been shown that the super-enhancer is required tostabilize the cell state or identity. In some embodiments, the cellstate is an embryonic-stem cell like state. Upon introduction of thesuper-enhancer into the cell, the super-enhancer drives expression ofgenes that are required to maintain the cell state or identityassociated with the super-enhancer.

In some aspects, cell state reflects the fact that cells of a particulartype can exhibit variability with regard to one or more features and/orcan exist in a variety of different conditions, while retaining thefeatures of their particular cell type and not gaining features thatwould cause them to be classified as a different cell type. Thedifferent states or conditions in which a cell can exist may becharacteristic of a particular cell type (e.g., they may involveproperties or characteristics exhibited only by that cell type and/orinvolve functions performed only or primarily by that cell type) or mayoccur in multiple different cell types. Sometimes a cell state reflectsthe capability of a cell to respond to a particular stimulus orenvironmental condition (e.g., whether or not the cell will respond, orthe type of response that will be elicited) or is a condition of thecell brought about by a stimulus or environmental condition. Cells indifferent cell states may be distinguished from one another in a varietyof ways. For example, they may express, produce, or secrete one or moredifferent genes, proteins, or other molecules (“markers”), exhibitdifferences in protein modifications such as phosphorylation,acetylation, etc., or may exhibit differences in appearance. Thus a cellstate may be a condition of the cell in which the cell expresses,produces, or secretes one or more markers, exhibits particular proteinmodification(s), has a particular appearance, and/or will or will notexhibit one or more biological response(s) to a stimulus orenvironmental condition. Markers can be assessed using methods wellknown in the art, e.g., gene expression can be assessed at the mRNAlevel using Northern blots, cDNA or oligonucleotide microarrays, orsequencing (e.g., RNA-Seq), or at the level of protein expression usingprotein microarrays, Western blots, flow cytometry,immunohistochemistry, etc. Modifications can be assessed, e.g., usingantibodies that are specific for a particular modified form of aprotein, e.g., phospho-specific antibodies, or mass spectrometry.

Another example of cell state is “activated” state as compared with“resting” or “non-activated” state. Many cell types in the body have thecapacity to respond to a stimulus by modifying their state to anactivated state. The particular alterations in state may differdepending on the cell type and/or the particular stimulus. A stimuluscould be any biological, chemical, or physical agent to which a cell maybe exposed. A stimulus could originate outside an organism (e.g., apathogen such as virus, bacteria, or fungi (or a component or productthereof such as a protein, carbohydrate, or nucleic acid, cell wallconstituent such as bacterial lipopolysaccharide, etc) or may beinternally generated (e.g., a cytokine, chemokine, growth factor, orhormone produced by other cells in the body or by the cell itself). Forexample, stimuli can include interleukins, interferons, or TNF alpha.Immune system cells, for example, can become activated upon encounteringforeign (or in some instances host cell) molecules. Cells of theadaptive immune system can become activated upon encountering a cognateantigen (e.g., containing an epitope specifically recognized by thecell's T cell or B cell receptor) and, optionally, appropriateco-stimulating signals. Activation can result in changes in geneexpression, production and/or secretion of molecules (e.g., cytokines,inflammatory mediators), and a variety of other changes that, forexample, aid in defense against pathogens but can, e.g., if excessive,prolonged, or directed against host cells or host cell molecules,contribute to diseases. Fibroblasts are another cell type that canbecome activated in response to a variety of stimuli (e.g., injury(e.g., trauma, surgery), exposure to certain compounds including avariety of pharmacological agents, radiation, etc.) leading them, forexample, to secrete extracellular matrix components. In the case ofresponse to injury, such ECM components can contribute to wound healing.However, fibroblast activation, e.g., if prolonged, inappropriate, orexcessive, can lead to a range of fibrotic conditions affecting diversetissues and organs (e.g., heart, kidney, liver, intestine, bloodvessels, skin) and/or contribute to cancer. The presence of abnormallylarge amounts of ECM components can result in decreased tissue and organfunction, e.g., by increasing stiffness and/or disrupting normalstructure and connectivity.

Another example of cell state reflects the condition of cell (e.g., amuscle cell or adipose cell) as either sensitive or resistant toinsulin. Insulin resistant cells exhibit decreased response tocirculating insulin; for example insulin-resistant skeletal muscle cellsexhibit markedly reduced insulin-stimulated glucose uptake and a varietyof other metabolic abnormalities that distinguish these cells from cellswith normal insulin sensitivity.

As used herein, a “cell state associated gene” is a gene the expressionof which is associated with or characteristic of a cell state ofinterest (and is often not associated with or is significantly lower inmany or most other cell states) and may at least in part be responsiblefor establishing and/or maintaining the cell state. For example,expression of the gene may be necessary or sufficient to cause the cellto enter or remain in a particular cell state.

In some aspects, modulating a super-enhancer function shifts a cell froman “abnormal” state towards a more “normal” state. In some embodiments,modulating a super-enhancer function shifts a cell from a“disease-associated” state towards a state that is not associated withdisease. A “disease-associated state” is a state that is typically foundin subjects suffering from a disease (and usually not found in subjectsnot suffering from the disease) and/or a state in which the cell isabnormal, unhealthy, or contributing to a disease.

In some embodiments, the methods and compounds herein are of use toreprogram a somatic cell, e.g., to a pluripotent state. In someembodiments the methods and compounds are of use to reprogram a somaticcell of a first cell type into a different cell type. In someembodiments, the methods and compounds herein are of use todifferentiate a pluripotent cell to a desired cell type.

In an embodiment, the method of modifying a cell state or identity canbe used to reprogram a cell to a less differentiated state, such methodcomprising the steps of: (a) contacting a differentiated cell orpopulation of cells with at least one reprogramming agent capable ofreprogramming said cell to less differentiated state; (b) maintainingsaid cell or population of cells under conditions appropriate forproliferation of said cell population and for activity of said at leastone reprogramming agent for a period of time sufficient to beginreprogramming of said cell or population of cells; and (c) transfectingsaid cell or population of cells with a nucleic acid constructcomprising a super-enhancer having a plurality of binding sites forcognate transcription factors Oct4, Sox2, and Nanog, whereintransfection of said cell drives high levels of expression of embryonicstem cell genes required to reprogram and maintain the cell in a lessdifferentiated state. In an embodiment, the less differentiated state isan embryonic stem cell-like state. Reprogramming of cells and suitablereprogramming agents (e.g., Oct4, Sox2, Nanog, etc.) are described infurther detail in U.S. Patent Application Publication No. 2011/0076678,U.S. Pat. No. 7,682,828, U.S. Pat. No. 8,071,369, U.S. PatentApplication Publication No. 2012/0028821, U.S. Patent Application Ser.No. 61/098,327, the teachings of all of which are incorporated herein byreference in their entirety.

In certain aspects, the present invention relates to a kit forreprogramming a differentiated somatic cell population to an embryonicstem-cell like state, comprising: (a) a population of differentiatedsomatic cells; (b) at least one reprogramming agent capable ofreprogramming said cell to an embryonic stem cell-like state; and (c) anucleic acid construct comprising a super-enhancer containing clustersof enhancers having binding sites for cognate transcription factorsOct4, Sox2, and Nanog; and (d) a reagent for transfecting saidpopulation of cells with said nucleic acid construct.

In some embodiments, modulating a function (activity) of asuper-enhancer is of use to treat, e.g., a metabolic, neurodegenerative,inflammatory, auto-immune, proliferative, infectious, cardiovascular,musculoskeletal, or other disease. It will be understood that diseasescan involve multiple pathologic processes and mechanisms and/or affectmultiple body systems. Discussion herein of a particular disease in thecontext of a particular pathologic process, mechanism, cell state, celltype, or affected organ, tissue, or system, should not be consideredlimiting. For example, a number of different tumors (e.g., hematologicneoplasms such as leukemias) arise from undifferentiated progenitorcells and/or are composed largely of undifferentiated or poorlydifferentiated cells that retain few if any distinctive featurescharacteristic of differentiated cell types. These tumors, which aresometimes termed undifferentiated or anaplastic tumors, may beparticularly aggressive and/or difficult to treat. In some embodimentsof the invention, a method of the invention is used to modify such cellsto a more differentiated state, which may be less highly proliferativeand/or more amenable to a variety of therapies, e.g., chemotherapeuticagents. In another embodiment, an inventive method is used to treatinsulin resistance which occurs, for example, in individuals sufferingfrom type II diabetes and pre-diabetic individuals. It would bebeneficial to modify the state of insulin-resistant cells towards a moreinsulin-sensitive state, e.g., for purposes of treating individuals whoare developing or have developed insulin resistance. In anotherembodiment, an inventive method is used to treat obesity.

Many inflammatory and/or autoimmune conditions may occur at least inpart as a result of excessive and/or inappropriate activation of immunesystem cells. Autoimmune diseases include, e.g., Graves disease,Hashimoto's thyroiditis, myasthenia gravis, rheumatoid arthritis,sarcoidosis, Sjögren's syndrome, scleroderma, ankylosing spondylitis,type I diabetes, vasculitis, and lupus erythematosus. Furthermore,immune-mediated rejection is a significant risk in organ and tissuetransplantation. Inflammation plays a role in a large number of diseasesand conditions. Inflammation can be acute (and may be recurrent) orchronic. In general, inflammation can affect almost any organ, tissue,or body system. For example, inflammation can affect the cardiovascularsystem (e.g., heart), musculoskeletal system, respiratory system (e.g.,bronchi, lungs), renal system, (e.g., kidneys), eyes, nervous system,gastrointestinal system (e.g., colon), integumentary system (e.g.,skin), musculoskeletal system (e.g., joints, muscles), resulting in awide variety of conditions and diseases. Chronic inflammation isincreasingly recognized as an important factor contributing toatherosclerosis and degenerative diseases of many types. Inflammationinfluences the microenvironment around tumours and contributes, e.g., totumor cell proliferation, survival and migration. Furthermore, chronicinflammation can eventually lead to fibrosis.

Exemplary inflammatory diseases include, e.g., adult respiratorydistress syndrome (ARDS), atherosclerosis (e.g., coronary arterydisease, cerebrovascular disease), allergies, asthma, cancer,demyleinating diseases, dermatomyositis, inflammatory bowel disease(e.g., Crohn's disease, ulcerative colitis), inflammatory myopathies,multiple sclerosis, glomerulonephritis, psoriasis, pancreatitis,rheumatoid arthritis, sepsis, vasculitis (including phlebitis andarteritis, e.g., polyarteritis nodosa, Wegener's granulomatosis,Buerger's disease, Takayasu's arteritis, etc.). In some embodiments, amethod of the invention is used to modify immune cell state to reduceactivation of immune system cells involved in such conditions and/orrender immune system cells tolerant to one or more antigens. In oneembodiment, dendritic cell state is altered. Promoting immune systemactivation using a method of the invention (e.g., in individuals whohave immunodeficiencies or have been treated with drugs that deplete ordamage immune system cells), potentially for limited periods of time,may be of benefit in the treatment of infectious diseases.

In other embodiments, activated fibroblasts are modified to a lessactivated cell state to reduce or inhibit fibrotic conditions or treatcancer.

Post-surgical adhesions can be a complication of, e.g., abdominal,gynecologic, orthopedic, and cardiothoracic surgeries. Adhesions areassociated with considerable morbidity and can be fatal. Development ofadhesions involves inflammatory and fibrotic processes. In someembodiments, a method of the invention is used to modify state of immunesystem cells and/or fibroblasts to prevent or reduce adhesion formationor maintenance.

In other embodiments, modifying cells to a more or less differentiatedstate is of use to generate a population of cells in vivo that aid inrepair or regeneration of a diseased or damaged organ or tissue, or togenerate a population of cells ex vivo that is then administered to asubject to aid in repair or regeneration of a diseased or damaged organor tissue.

In some embodiments, cell type and/or cell state becomes modified overthe course of multiple cell cycle(s). In some embodiments, cell typeand/or cell state is stably modified. In some embodiments, a modifiedtype or state may persist for varying periods of time (e.g., days,weeks, months, or indefinitely) after the cell is no longer exposed tothe agent(s) that caused the modification. In some embodiments,continued or at intermittent exposure to the agent(s) is required orhelpful to maintain the modified state or type.

Cells may be in living animal, e.g., a mammal, or may be isolated cells.Isolated cells may be primary cells, such as those recently isolatedfrom an animal (e.g., cells that have undergone none or only a fewpopulation doublings and/or passages following isolation), or may be acell of a cell line that is capable of prolonged proliferation inculture (e.g., for longer than 3 months) or indefinite proliferation inculture (immortalized cells). In many embodiments, a cell is a somaticcell. Somatic cells may be obtained from an individual, e.g., a human,and cultured according to standard cell culture protocols known to thoseof ordinary skill in the art. Cells may be obtained from surgicalspecimens, tissue or cell biopsies, etc. Cells may be obtained from anyorgan or tissue of interest. In some embodiments, cells are obtainedfrom skin, lung, cartilage, breast, blood, blood vessel (e.g., artery orvein), fat, pancreas, liver, muscle, gastrointestinal tract, heart,bladder, kidney, urethra, prostate gland. Cells may be maintained incell culture following their isolation. In certain embodiments, thecells are passaged or allowed to double once or more following theirisolation from the individual (e.g., between 2-5, 5-10, 10-20, 20-50,50-100 times, or more) prior to their use in a method of the invention.They may be frozen and subsequently thawed prior to use. In someembodiments, the cells will have been passaged or permitted to double nomore than 1, 2, 5, 10, 20, or 50 times following their isolation fromthe individual prior to their use in a method of the invention. Cellsmay be genetically modified or not genetically modified in variousembodiments of the invention. Cells may be obtained from normal ordiseased tissue. In some embodiments, cells are obtained from a donor,and their state or type is modified ex vivo using a method of theinvention. The modified cells are administered to a recipient, e.g., forcell therapy purposes. In some embodiments, the cells are obtained fromthe individual to whom they are subsequently administered.

A population of isolated cells in any embodiment of the invention may becomposed mainly or essentially entirely of a particular cell type or ofcells in a particular state. In some embodiments, an isolated populationof cells consists of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100% cells of a particular type or state (i.e.,the population is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99%, or 100% pure), e.g., as determined by expression of oneor more markers or any other suitable method.

In certain aspects, the present invention relates to a method ofselectively inhibiting expression of an aberrantly expressed genecomprising disrupting the function of a super-enhancer associated withthe gene. In certain embodiments, the gene comprises an oncogene. Duringthe course of work described herein, the present inventors have observedthat disruption of super-enhancers by BRD4 inhibition led to a dramaticloss of expression of critical tumor genes, accompanied by a potentanti-proliferative effect. Given the fact that super-enhancers arecommon features of mammalian cells, and that super-enhancers have beenshown to drive high levels of gene expression, it is reasonable toexpect that super-enhancer disruption can be used to selectively inhibitexpression of any gene (e.g., any gene that is overexpressed in adiseased cell, wherein the gene is associated with a super-enhancer) bydisrupting the super-enhancer associated with the oncogene. In anembodiment, the oncogene is MYC. In an embodiment, the oncogene is IRF4.

It should be appreciated that the present invention contemplates the useof any technique or any agent that is capable of disrupting the functionof the super-enhancer. Generally, disrupting the function of thesuper-enhancer involves contacting said super-enhancer region with aneffective amount of an agent that interferes with occupancy of thesuper-enhancer region by a cognate transcription factor for the gene, atranscriptional coactivator, or a chromatin regulator. In someembodiments, disrupting the function of the super-enhancer can beachieved by contacting the super-enhancer region with a pause releaseagent. In certain embodiments, the agent interferes with a binding siteon the super-enhancer for the cognate transcription factor, interfereswith interaction between the cognate transcription factor and atranscriptional coactivator, inhibits the transcription coactivator, orinterferes with or inhibits the chromatin regulator. In someembodiments, the agent is a bromodomain inhibitor. In some embodiments,the agent is a BRD4 inhibitor. In some embodiments, the agent is thecompound JQ1. In some embodiments, the agent is iBET.

Any of a wide variety of agents (also termed “compounds”) can be used todisrupt the function of the super-enhancer, such as BET bromodomaininhibitors, P-TEFb inhibitors or compounds that interfere with bindingof the cognate transcription factors to the binding sites of thesuper-enhancer associated with the gene (e.g, if the gene is anoncogene, such as MYC, a c-Myc inhibitor can be used to disrupt thefunction of the super-enhancer). An inhibitor could be any compoundthat, when contacted with a cell, results in decreased functionalactivity of a molecule or complex, e.g., transcriptional coactivator(e.g., Mediator), a chromatin regulator (e.g., BRD4), an elongationfactor (e.g., P-TEFb), or cognate transcription factor (e.g., a cognateoncogenic transcription factor), in the cell. An inhibitor could actdirectly, e.g., by physically interacting with a molecule or complex tobe inhibited, or a component thereof, or indirectly such as byinteracting with a different molecule or complex required for activityof the molecule or complex to be inhibited, or by interfering withexpression or localization.

Compounds of use in various embodiments of the invention can comprise,e.g., small molecules, peptides, polypeptides, nucleic acids,oligonucleotides, etc. Certain non-limiting examples are presentedbelow.

A small molecule is often an organic compound having a molecular weightequal to or less than 2.0 kD, e.g., equal to or less than 1.5 kD, e.g.,equal to or less than 1 kD, e.g., equal to or less than 500 daltons andusually multiple carbon-carbon bonds. Small molecules often comprise oneor more functional groups that mediate structural interactions withproteins, e.g., hydrogen bonding, and typically include at least anamine, carbonyl, hydroxyl or carboxyl group, and in some embodiments atleast two of the functional chemical groups. A small molecule maycomprise cyclic carbon or heterocyclic structures and/or aromatic orpolyaromatic structures substituted with one or more chemical functionalgroups and/or heteroatoms. In some embodiments a small moleculesatisfies at least 3, 4, or all criteria of Lipinski's “Rule of Five”.In some embodiments, a compound is cell-permeable, e.g., within therange of typical compounds that act intracellularly, e.g., withinmammalian cells. In some embodiments, the IC50 of a compound, e.g., asmall molecule, for a target to be inhibited is less than or equal toabout 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 μM, 10 μM, 50 μM, or 100 μM,

Nucleic acids, e.g., oligonucleotides (which typically refers to shortnucleic acids, e.g., 50 nucleotides in length or less), the inventioncontemplates use of oligonucleotides that are single-stranded,double-stranded (ds), blunt-ended, or double-stranded with overhangs, invarious embodiments of the invention. The full spectrum of modifications(e.g., nucleoside and/or backbone modifications), non-standardnucleotides, delivery vehicles and systems, etc., known in the art asbeing useful in the context of siRNA or antisense-based molecules forresearch or therapeutic purposes is contemplated for use in variousembodiments of the instant invention. In some embodiments a compound isan RNAi agent, antisense oligonucleotide, or aptamer. The term “RNAiagent” encompasses nucleic acids that can be used to achieve RNAsilencing in mammalian cells. As used herein RNA silencing, also termedRNA interference (RNAi), encompasses processes in whichsequence-specific silencing of gene expression is effected by anRNA-induced silencing complex (RISC) that has a short RNA strandincorporated therein, which strand directs or “guides” sequence-specificdegradation or translational repression of mRNA to which it hascomplementarity. The complementarity between the short RNA and mRNA neednot be perfect (100%) but need only be sufficient to result ininhibition of gene expression. For example, the degree ofcomplementarity and/or the characteristics of the structure formed byhybridization of the mRNA and the short RNA strand can be such that thestrand can (i) guide cleavage of the mRNA in the RNA-induced silencingcomplex (RISC) and/or (ii) cause translational repression of the mRNA byRISC. The short RNA is often incorporated into RISC as part of a shortdouble-stranded RNA (dsRNA), RNAi may be achieved artificially ineukaryotic, e.g., mammalian, cells in a variety of ways. For example,RNAi may be achieved by introducing an appropriate short double-strandednucleic acid into the cells or expressing in the cells a nucleic acidthat is processed intracellularly to yield such short dsRNA. ExemplaryRNAi agents are a short hairpin RNA (shRNA), a short interfering RNA(siRNA), micrRNA (miRNA) and a miRNA precursor. siRNAs typicallycomprise two separate nucleic acid strands that are hybridized to eachother to form a duplex. They can be synthesized in vitro, e.g., usingstandard nucleic acid synthesis techniques. A nucleic acid may containone or more non-standard nucleotides, modified nucleosides (e.g., havingmodified bases and/or sugars) or nucleotide analogs, and/or have amodified backbone. Any modification or analog recognized in the art asbeing useful for RNAi, aptamers, antisense molecules or other uses ofoligonucleotides can be used. Some modifications result in increasedstability, cell uptake, potency, etc. Exemplary compound can comprisemorpholinos or locked nucleic acids. In some embodiments the nucleicacid differs from standard RNA or DNA by having partial or complete2′-0-methylation or 2′-0-methoxyethyl modification of sugar,phosphorothioate backbone, and/or a cholesterol-moiety at the 3′-end. Incertain embodiments the siRNA or shRNA comprises a duplex about 19nucleotides in length, wherein one or both strands has a 3′ overhang of1-5 nucleotides in length (e.g., 2 nucleotides), which may be composedof deoxyribonucleotides. shRNA comprise a single nucleic acid strandthat contains two complementary portions separated by a predominantlynon-self-complementary region. The complementary portions hybridize toform a duplex structure and the non-self-complementary region forms aloop connecting the 3′ end of one strand of the duplex and the 5′ end ofthe other strand. shRNAs can undergo intracellular processing togenerate siRNAs. In certain embodiments the term “RNAi agent” alsoencompasses vectors, e.g., expression vectors, that comprise templatesfor transcription of an siRNA (e.g., as two separate strands that canhybridize), shRNA, or microRNA precursor, and can be used to introducesuch template into mammalian cells and result in transient or stableexpression thereof.

In some embodiments an RNAi agent, aptamer, antisense oligonucleotide,other nucleic acid, peptide, polypeptide, or small molecule isphysically associated with a moiety that increases cell uptake, such asa cell-penetrating peptide, or a delivery agent. In some embodiments adelivery agent at least in part protects the compound from degradation,metabolism, or elimination from the body (e.g., increases thehalf-life). A variety of compositions and methods can be used to deliveragents to cells in vitro or in vivo. For example, compounds can beattached to a polyalkylene oxide, e.g., polyethylene glycol (PEG) or aderivative thereof, or incorporated into or attached to various types ofmolecules or particles such as liposomes, lipoplexes, or polymer-basedparticles, e.g., microparticles or nanoparticles composed at least inpart of one or more biocompatible polymers or copolymers comprisingpoly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesterardes, polyorthoesters, polyhydroxybutyric acid, and/or polyanhydrides.

In some embodiments, an agent comprises a polypeptide. A “polypeptide”refers to a polymer of amino acids linked by peptide bonds. A protein isa molecule comprising one or more polypeptides. A peptide is arelatively short polypeptide, typically between about 2 and 100 aminoacids (aa) in length, e.g., between 4 and 60 aa; between 8 and 40 aa;between 10 and 30 aa. The terms “protein”, “polypeptide”, and “peptide”may be used interchangeably. In general, a polypeptide may contain onlystandard amino acids or may comprise one or more non-standard aminoacids (which may be naturally occurring or non-naturally occurring aminoacids) and/or amino acid analogs in various embodiments. A “standardamino acid” is any of the 20 L-amino acids that are commonly utilized inthe synthesis of proteins by mammals and are encoded by the geneticcode. A “non-standard amino acid” is an amino acid that is not commonlyutilized in the synthesis of proteins by mammals. Non-standard aminoacids include naturally occurring amino acids (other than the 20standard amino acids) and non-naturally occurring amino acids. In someembodiments, a non-standard, naturally occurring amino acid is found inmammals. For example, ornithine, citrulline, and homocysteine arenaturally occurring non-standard amino acids that have important rolesin mammalian metabolism. Exemplary non-standard amino acids include,e.g., singly or multiply halogenated (e.g., fluorinated) amino acids,D-amino acids, homo-amino acids, N-alkyl amino acids (other thanproline), dehydroamino acids, aromatic amino acids (other thanhistidine, phenylalanine, tyrosine and tryptophan), and α,αdisubstituted amino acids. An amino acid, e.g., one or more of the aminoacids in a polypeptide, may be modified, for example, by addition, e.g.,covalent linkage, of a moiety such as an alkyl group, an alkanoyl group,a carbohydrate group, a phosphate group, a lipid, a polysaccharide, ahalogen, a linker for conjugation, a protecting group, etc.Modifications may occur anywhere in a polypeptide, e.g., the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.A given polypeptide may contain many types of modifications.Polypeptides may be branched or they may be cyclic, with or withoutbranching. Polypeptides may be conjugated with, encapsulated by, orembedded within a polymer or polymeric matrix, dendrimer, nanoparticle,microparticle, liposome, or the like. Modification may occur prior to orafter an amino acid is incorporated into a polypeptide in variousembodiments. Polypeptides may, for example, be purified from naturalsources, produced in vitro or in vivo in suitable expression systemsusing recombinant DNA technology (e.g., by recombinant host cells or intransgenic animals or plants), synthesized through chemical means suchas conventional solid phase peptide synthesis, and/or methods involvingchemical ligation of synthesized peptides (see, e.g., Kent, S., J PeptSci., 9(9):574-93, 2003 or U.S. Pub. No. 20040115774), or anycombination of the foregoing.

One of ordinary skill in the art will understand that a protein may becomposed of a single amino acid chain or multiple chains associatedcovalently or noncovalently. In some embodiments, the agent is anon-functional mutant of the cognate oncogenic transcription factor, thetranscriptional coactivator, or the chromatin regulator that mimicsinteractions of the cognate oncogenic transcription factor, thetranscriptional coactivator, or the chromatin regulator but lacks theability to activate transcription of the oncogene. For example, apolypeptide can be a dominant negative version of Mediator, anelongation factor (e.g., P-TEFb subunit) or a dominant negative versionof a cognate oncogenic transcription factor (e.g., a c-Myc or Max). Apolypeptide that binds to and inhibits Mediator or P-TEFb or c-Myc couldbe identified, e.g., using phage display.

In some embodiments a compound comprises an antibody. The term“antibody” encompasses immunoglobulins and derivatives thereofcontaining an immunoglobulin domain capable of binding to an antigen. Anantibody can originate from any mammalian or avian species, e.g., human,rodent (e.g., mouse, rabbit), goat, chicken, etc., or can be generatedusing, e.g., phage display. The antibody may be a member of anyimmunoglobulin class, e.g., IgG, IgM, IgA, IgD, IgE, or subclassesthereof such as IgG1, IgG2, etc. In various embodiments of the invention“antibody” refers to an antibody fragment such as an Fab′, F(ab′)2, scFv(single-chain variable) or other fragment that retains an antigenbinding site, or a recombinantly produced scFv fragment, includingrecombinantly produced fragments. An antibody can be monovalent,bivalent or multivalent in various embodiments. The antibody may be achimeric or “humanized” antibody, which can be generated using methodsknown in the art. An antibody may be polyclonal or monoclonal, thoughmonoclonal antibodies may be preferred. Methods for producing antibodiesthat specifically bind to virtually any molecule of interest are knownin the art. In some aspects the antibody is an intrabody, which may beexpressed intracellularly. In some embodiments a compound comprises asingle-chain antibody and a protein transduction domain (e.g., as afusion polypeptide).

In some embodiments, a composition or method of the invention employs atranscriptional coactivator inhibitor, a chromatin regulator inhibitor,an elongation factor or pause release inhibitor, or a cognatetranscription factor inhibitor that are small molecules.

In some embodiments, the agent is a BET bromodomain inhibitor. In someembodiments, the agent is a BRD4 inhibitor. In some embodiments, theagent is JQ1. In some embodiments, the agent is iBET. In someembodiments, the elongation factor or pause release inhibitor is aP-TEFb inhibitor. In some embodiments, the cognate oncogenictranscription factor inhibitor is a c-Myc inhibitor. In someembodiments, a composition or method employs a Mediator inhibitor, aBRD4 inhibitor, a P-TEFb inhibitor and a c-Myc inhibitor that eachcomprise a nucleic acid, e.g., RNAi agents. In some embodiments, acomposition or method employs a Mediator inhibitor, a P-TEFb inhibitorthat comprises a nucleic acid, e.g., RNAi agents, e.g., siRNAs. In someembodiments, the Mediator inhibitor may bind to a Mediator component,Mediator complex, or a Mediator associated protein, for example, anantibody directed against the Mediator component, Mediator complex, orthe Mediator associated protein. Examples of suitable antibodies can befound in PCT International Application No. WO 2011/100374, the teachingsof which are incorporated herein by reference in their entirety.

In some embodiments the material is isolated using an agent (e.g., anantibody) that binds to a Mediator component, Mediator complex, or thatbinds to a Mediator-associated protein.

In some embodiments, the agent is a nucleic acid that hybridizes to abinding site on the super-enhancer for the cognate transcription factor.

Compounds can be produced using any suitable method known in the art.The skilled artisan will select an appropriate method based, e.g., onthe nature of the compound. The production method can be partially orcompletely synthetic in various embodiments. In some embodiments acompound (or starting material for synthesis) is purified from anorganism or other natural source, e.g., a plant, microbe, fermentationbroth, etc. A compound of use in the invention may be provided as partof a composition, which may contain, e.g., an ion, salt, aqueous ornon-aqueous diluent or carrier, buffer, preservative, etc. It is notedthat although combined use of compounds is of particular interest, theuse of compounds disclosed herein is not limited to their use incombination. In some embodiments of the invention, a compound may beused as a single agent.

In some embodiments, a P-TEFb inhibitor inhibits CDK9 kinase activity.The compound may inhibit one or more additional kinases, e.g., CDKs, inaddition to CDK9. Often a kinase inhibitor acts by binding to an ATPbinding pocket of a kinase. Thus in some embodiments a CDK9 inhibitorbinds to the ATP binding pocket of CDK9. In some embodiments the P-TEFbinhibitor is selective for CDKs relative to many, most, or all otherkinase families. In some embodiments the CDK inhibitor is selective forCDKs 1, 4, and 9 versus CDK2. In some embodiments the P-TEFb inhibitoris a CDK inhibitor that is selective for CDK9 versus CDK2. In someembodiments the P-TEFb inhibitor is a CDK inhibitor that is selectivefor CDK9 versus CDK1 and CDK4. It will be appreciated that kinaseinhibitory activity is tested against CKDs in complex with a preferredcyclin partner. For example, in some embodiments CDK2 activity can betested using cyclin A. It will also be appreciated that a kinase assaycan employ a relevant substrate, e.g., a physiologically relevantsubstrate or portion thereof comprising a phosphoryation site for thekinase.

In some embodiments, the compound is an N-methylpiperidinyl,chlorophenyl flavone. In some embodiments, the compound is flavopiridolor a flavopiridol analog.

Flavopiridol(−)-2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methyl-4-piperidinyl]-4H-1-benzopyran-4-onehydrochloride is a synthetic flavone that inhibits multiple CDKs,including CDK9. Its structure is shown below.

Flavopiridol has been shown to have antitumor activity against varioustumor cells lines and to inhibit tumor growth in xenograft models. Ithas undergone clinical trials in a number of different cancer typesincluding various solid tumors and leukemias. As described further inthe examples, flavopiridol was shown to inhibit pause release. Withoutwishing to be bound by theory, this may help counteract the effects ofMyc overexpression, and this may be the basis for the therapeutic effectof flavopiridol on some tumors.

Flavopiridol analogs include compounds designed based on flavopiridol,e.g., by modifying one or more of the rings of the flavopiridolstructure at one or more positions. In some embodiments, a flavopiridolanalog is a 2-thio or 2-oxo flavopiridol analog. For example, PCT/US1997/007610 describes compounds of formula I:

wherein X is oxygen or sulfur; R¹, R², R³, R⁴, R⁵, R⁶, m, and n are asdefined in PCT/US1997/007610.

Additional flavopiridol analogs are disclosed in Murthi, K. K., et al.,Bioorg Med Chem Lett. 10(10): 1037-41, 2000, which describesmodifications of the 3-hydroxy-1-methylpiperidinyl (D ring) offlavopiridol.

In some embodiments, a flavopiridol analog has the following structure:

In some embodiments R is phenyl or substituted phenyl, e.g., halogenatedphenyl. In some embodiments, R is selected from the group consisting of:3-chlorophenyl, 4-chlorophenyl, 2-fluorophenyl, 4-fluorophenyl,4-bromophenyl, 4-t-butylphenyl, 4-trifluoromethylphenyl,4-hydroxyphenyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 4-chloro-3-pyridyl,5-methylisoxazole, 3-vinylphenyl, 4-vinylphenyl, 2-chlorophenyl,4-fluorophenyl, 2-bromophenyl, and 3-pyridyl. In some embodiments thecompound displays increased selectivity for CDK9 than does flavopiridol.See, e.g., Ali, A., et al., Chembiochem, 10(12):2072-80, 2009, foradditional information regarding these compound.

In some embodiments, a CDK9 inhibitor has the following structure:

wherein R1, R2, R3, R4, and R9 are as defined in PCT/IB 2006/052002(WO/2007/148158). In some embodiments (i) R1 comprises an aromaticgroup; (ii) R4 comprises an R—(OH) group, wherein R is a C₁₋₆ aliphaticgroup; (iii) R9 comprises a C₁₋₆ aliphatic group, e.g, a methyl group;or (iv) any combination of (i), (ii), and (iii). In some embodiments,the compound may have the following structure:

wherein R comprises an aromatic group.

Crystal structures of P-TEFb (CDK9/cyclin T1) alone and in a complexwith flavopiridol are available (Baumli, S., et al., EMBO J. 27(13):1907-18, 2008). Flavopiridol was shown to bind to the ATP binding pocketof CDK9. Structural information can be used in the design of additionalP-TEFb inhibitors including, but not limited to, additional analogs offlavopiridol. Furthermore, virtual screening can be performed usingstructural information regarding diverse chemical compounds to identifycandidate P-TEFb inhibitors. In some embodiments, a P-TEFb inhibitor isa compound that makes similar intermolecular contacts with CDK9 as doesflavopiridol. Similar approaches can be used to design analogs of otherCDK9 inhibitors.

In some embodiments, a flavopiridol analog exhibiting reduced binding tohuman serum relative to flavopiridol is used.

In some embodiments, the P-TEFb inhibitor is a purine or purine analog,e.g., a biaryl purine analog. In some embodiments, the purine analog isa 2,6,9-substituted purine analog. In some embodiments, the compound isroscovitine, e.g., S-roscovitine or R-roscovitine. Unless otherwiseindicated, where roscovitine is mentioned herein, the roscovitine can beR-roscovitine (also called Selicielib or CYC202;2-(R)-(l-Ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropyl purine).Roscovitine is a CDK inhibitor that preferentially inhibit multipleenzyme targets including CDK1, CDK2, CDK7 and CDK9 and has been studiedin clinical trials for treatment of a variety of proliferative diseases.

In some embodiments the compound is a roscovatine analog. Exemplaryroscovitine analogs are oloumicine(2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine), olomoucine II(6-[(2-hydroxybenzyl)amino]-2-[[1-(hydroxymethyl)propyl]amino]-9-isopropylpurine)and LGR1406(N-5-(2-aminocyclohexyl)-N-7-benzyl-3-isopropyl-1(2)H-pyrazolo[4,3-d]pyrimidine-5,7-di-amine).Roscovitine analogs generated by introduction of an aryl ring onto the4-position of the C-6 benzyl amino group of roscovitine, and a series ofC-6 biarylmethylamino derivatives prepared with modifications on the C-6biaryl rings, N-9 and C-2 positions, are described in Trova, M P, et.al., Bioorg Med Chem Lett. 19(23):6608-12, 2009.

Many additional CDK inhibitors are known in the art that may inhibitCDK9, optionally with at least some selectivity relative to inhibitionof one or more other CDKs. For example, PCT/US2009/049637(WO/2010/003133) discloses compounds that are

reported to inhibit CDK9. In some aspects, the compounds have thefollowing structure, where R1 and R3 are as defined therein.

PCT/EP2008/063715 (WO 2009047359) discloses additional compounds thatare reported to inhibit CDK9. In some aspects, the compounds have thefollowing structure, wherein R1, R2, Ra, and (R3)_(x) are as definedtherein.

In some embodiments, a P-TEFb inhibitor comprises an RNAi agent (e.g.,an siRNA) or an antisense oligonucleotide that inhibits expression of aP-TEFb subunit (e.g., CDK9, cyclin T1, T2a, T2b, or K). In someembodiments a P-TEFb inhibitor comprises an antibody or aptamer thatspecifically binds to a P-TEFb subunit. Optionally the antibody oraptamer may bind to multiple CDKs or cyclins.

In some embodiments, a c-Myc inhibitor is a small molecule. In someembodiments, a c-Myc inhibitor inhibits formation of c-Myc/Maxheterodimers. In some embodiments, a c-Myc inhibitor inhibits binding ofc-Myc/Max to a target site in DNA. In some embodiments a c-Myc inhibitoris relatively specific for inhibiting transcription mediated by c-Mycrelative to transcription mediated by many or most other basichelix-loop-helix/leucine zipper transcription factors.

Various compounds that inhibit c-Myc are described in Berg, T., Curr.Op. Chem. Biol., 12: 464-471, 2008, and references therein. The peptidemimetic IIA6B17 is described in Berg, T., et al., Proc Natl Acad Sci USA99 (2002), pp. 3830-3835 and was shown to inhibit c-Myc-dependenttranscription in a reporter gene assay (X. Lu, et al. Oncol Rep 19(2008), pp. 825-830). Testing a 285 member chemical library derived fromplanar, aromatic scaffolds in a c-Myc/Max dimerization assay led toidentification of four structurally related Myc/Max dimerizationinhibitors, which also inhibited DNA binding of c-Myc/Max (Y. Xu, et al.Bioorg Med Chem 14 (2006), pp. 2660-2673.) For example, the compoundNY2267 strongly inhibited c-Myc-dependent oncogenic transformation ofchicken embryo fibroblasts at 20 μM, showed selectivity overtransformation mediated by v-Src or v-Jun, but did not discriminatebetween transcription mediated by c-Jun and c-Myc. Several compoundswere selected from a chemical library on the basis of their ability toprevent association of the HLH-Zip domains of c-Myc and Max in a yeasttwo-hybrid assay (X. Yin, et al., Oncogene 22 (2003), pp. 6151-6159).One, 10058-F4 (IC₅₀=49 μM on HL60 cells), served as starting point forthe testing of derivatives with improved activities. One of the numerousderivatives resulting from structural variation of the substituents onthe aromatic ring and the rhodanine moiety, the compound 28RH-NCN-1,inhibited DNA binding of c-Myc with activity comparable to that of theparent compound, and inhibited growth of HL60 cells with improvedpotency (IC₅₀=29 μM) (Wang, H., et al., Mol Cancer Ther 6 (2007), pp.2399-2408). See also PCT/US2007/004039 (WO/2007/098010).

Screening chemical libraries for compounds that inhibited DNA binding ofc-Myc, led to discovery of the pyrazolo[1,5-a]pyrimidine Mycro1(Kiessling, A., et al., Chem Biol 13 (2006), pp. 745-751). Mycro1 andthe derivative Mycro2 were subsequently shown to inhibit c-Myc/Maxdimerization, c-Myc-dependent proliferation, gene transcription, andoncogenic transformation. While Mycro1 and Mycro2 displayed goodspecificities in vitro, they showed only weak-to-moderate specificityfor c-Myc-dependent transcription over transcription mediated by AP-1family proteins, which also dimerize via leucine zippers. A follow-upscreen using a focused library of pyrazolo[1,5-a]pyrimidines led to thediscovery of the pyrazolo[1,5-f1]pyrimidine 1 (Mycro3), which inhibitedc-Myc/Max dimerization and DNA binding with very good selectivity invitro, and also showed good potency and selectivity at concentrations of10-40 μM against c-Myc in cellular assays (A. Kiessling, A, et al.,ChemMedChem 2 (2007), pp. 627-630).

It can be reasoned that inhibitors of the DNA-protein interactionsbetween intact c-Myc/Max dimers and their DNA recognition motif shouldnot interfere with gene transcription repressed by c-Myc, but wouldstill block c-Myc induced transcriptional activation. This distinctioncan be used to help selectively identify compounds having this mechanismof action. In a screen designed to identify compounds that particularlyaffect cells with high levels of c-Myc, a compound termed MYRA-A, wasdiscovered, which was shown to inhibit Myc-regulated gene expression,oncogenic transformation, and to induce apoptosis in a Myc-dependentmanner (H. Mo and M. Hennksson, Proc Natl Acad Sci USA 103 (2006), pp.6344-6349). In a subsequent study, the same group published anadditional inhibitor of DNA binding of c-Myc/Max family members dubbedNSC308848 (Mo, H., et al. Cell Cycle 5 (2006), pp. 2191-2194).Hammoudeh, et al. (2009) identified multiple small molecule bindingsites on c-Myc, facilitating use of drug design and/or virtual screeningto identify additional c-Myc inhibitors.

Some exemplary small molecule c-Myc inhibitors of use in variousembodiments of the invention are shown below. In certain embodiments ofthe invention analogs of any of these compounds are used.

In some embodiments, a c-Myc inhibitor comprises an RNAi agent (e.g., ansiRNA) or an antisense oligonucleotide that inhibits expression ofc-Myc. In some embodiments a c-Myc inhibitor comprises an antibody oraptamer that specifically binds to c-Myc.

In some embodiments the agent promotes proteolysis of a polypeptideencoded by an oncogene in a cell (e.g., a tumor cell) exhibitingexcessive levels of the cognate transcription factor and moretranscriptional coactivator and chromatin regulator occupancy of thesuper-enhancer then the average single enhancer for the oncogene (e.g.,an order of magnitude more). In some embodiments the agent promotesglobal proteolysis in cell-specific manner such that global proteolysisis only induced in those cells (e.g., tumor cells) exhibiting extremelyhigh levels of the cognate transcription factor of the gene andtranscriptional coactivator super-enhancer occupancy. In someembodiments the agent promotes proteolysis of a polypeptide encoded byone or more of a plurality of oncogenes in a cell in which cognatetranscription factor levels are high and super-enhancers of the oncogeneare occupied by more transcriptional coactivator than the average singleenhancer of the oncogene.

The present invention contemplates the use of any agent that is capableof promoting proteolysis. In some embodiments the agent promotes globalproteolysis of polypeptides encoded by the oncogenes. In someembodiments the agent promotes global proteolysis of polypeptidesencoded by the oncogenes is promoted in cells that exhibit elevatedcognate oncogenic transcription factors for the oncogene. In someembodiments the agent promotes global proteolysis of polypeptides isspecific to tumor cells that possess oncogenes associated withsuper-enhancers. In some embodiments the agent promotes globalproteolysis of polypeptides in cells that exhibit elevated cognateoncogenic transcription factors and excessive levels of transcriptionalco-activator and/or chromatin regulator co-occupancy of super-enhancersand active transcription start sites.

In some embodiments the agent promotes global proteolysis ofpolypeptides by targeting the oncogene and its expression products forubiquitin-dependent proteolysis. In some embodiments, the agent promotesglobal proteolysis of polypeptides by ubiquitin-dependent proteolysis bythe proteasome. Ubiquitin-dependent proteolysis is a pathway used byeukaryotic cells for degrading cellular proteins. Protein ubiquitinationis catalyzed by the concerted actions of three classes of enzymes; theE1 ubiquitin-activating enzymes, the E2 ubiquitin-conjugating enzymes,and the E3 ubiquitin protein ligases (Hochstrasser, Annu Rev. Genet 30:405-39, 1996). E1 and E2 are involved in the activation and transfer ofubiquitin, while the substrate specificity of the ubiquitin pathway isconferred by the E3 ubiquitin protein ligases. In some embodiments theagent comprises a ubiquitin protein ligase polypeptide. In someembodiments the agent is an E3 ubiquitin protein ligase polypeptide. Insome embodiments the E3 ubiquitin protein ligase is an SCF polypeptide.In some embodiments the agent is a HECT polypeptide. In some embodimentsthe agent is a UBRI polypeptide. In some embodiments the E3 ubiquitinprotein ligase polypeptide is an F-box polypeptide (e.g., an F-boxpolypeptide which further comprises a WD domain). In some embodimentsthe F-box polypeptide is Cdc4p. In some embodiments the F-boxpolypeptide is Pop1p. In some embodiments the F-box polypeptide is Pop2p. In some embodiments the F-box polypeptide is Grr1p. In someembodiments the F-box polypeptide is Met30p. In some embodiments theF-box polypeptide is HOSp. In some embodiments the F-box polypeptide isbeta TrCPp. In some embodiments the F-box polypeptide is FWD1p. In someembodiments the F-box polypeptide is a polypeptide which is at least 70%identical to a contiguous polypeptide sequence of a polypeptide selectedfrom the group consisting of SEQ ID Nos. 2, 4, 6, 8, 10, and 12described in U.S. Pat. No. 7,223,556, which is incorporated herein byreference. In some embodiments the F-box polypeptide is at least 80%identical to a contiguous nucleic acid sequence of SEQ ID Nos. 1, 3, 5,7, 9, and 11 described in U.S. Pat. No. 7,223,556, which is incorporatedherein by reference.

In some embodiments the agent destabilizes RNA and/or proteins producedby the oncogene. In some embodiments an agent that destabilizes RNA isan agent that modulates nonsense-mediated RNA decay (NMD). Gardnerdiscusses NMD implications for tumorigenesis (Gardner. Mol Cancer Res.8; 295, 2010). In some embodiments an agent that modulates NMD is anagent that induces NMD of RNA transcripts of cognate oncogenictranscription factors, transcriptional coactivators, or chromatinregulators. In some embodiments an agent that modulates NMD is an agentthat downregulates NMD that has been upregulated in a tumor. In someembodiments an agent that modulates NMD is an agent that inhibits Upf1.In some embodiments an agent that inhibits Upf1 is Pateamine A (PatA),as is described by Dang et al. (Dang et al. J Biol Chem.284(35):23613-21, 2009).

In some embodiments the agent blocks mRNA splicing. In some embodimentsan agent that blocks mRNA splicing interferes with alternative splicing.In some embodiments an agent that blocks mRNA splicing is a specificinhibitor of CDC2-like kinase isoforms 1 and 4 (CLK1/CLK4) known asKH-CB19, as is described in Fedorov et al. (Fedorov et al. Chem Biol.18(1):67-76, 2011). In some embodiments an agent that interferes withalternative spicing is amiloride, as is described by Chang et al. PLosONE. 6(6):e18643).

In some embodiments an agent that blocks mRNA splicing is an inhibitorof spliceosome catalysis. In some embodiments an agent that inhibitsspliceosome catalysis is a 1,4-napthoquinones and/or a 1,4-heterocyclicquinone, non-limiting examples of which are described by Berg et al.(Berg et al. Mol Cell Biol. 32(7):1271-83, 2012). In some embodimentsthe splicing inhibitor comprises the benzothiazole-4,7-dione, BN82685,which blocks the second of two trans-esterification splicing reactions,preventing the release of intron lariat and exon ligation (Berg et al.2012). In an embodiment an agent that blocks mRNA splicing comprises4μ8C, which blocks substrate access to an IRE1 active site andselectively inactivates Xpb1 splicing, as is described by Cross et al.(Cross et al. Proc Natl Acad Sci USA, Epub ahead of print on Feb. 6,2012).

In some embodiments the agent inhibits translation of mRNA into protein.In some embodiments an agent that inhibits translation of mRNA intoprotein comprises a nucleoside 5′-monophosphate analog of the mRNA5′-cap, for example, Barzynkiewics et al. describe nucleotide capanalogs of 7-methylguanosine 5′monophosphate (m7GMP) that acted ascompetitive inhibitors of capped mRNA translation, including analogs inwhich the 7-methyl moiety is substituted with 7-ethyl (e7), 7-propyl(p7), 7-isopropyl (ip7), 7-butyl (b7), 7-isobutyl (ib7), 7-cyclopentyl(cp7), 7-(carboxymethyl) (cm7), 7-benzyle (bn7), 7-(2-phenylethyi)[7-(2-PhEt)], and 7-(1-penylethyl) [7-(1-PhEt)]. (Darzynkiewics et al.28(11):4771-8, 1989).

It should be appreciated that the various agents described herein can beused alone, or in combination with other agents described, for example,an agent that interferes with c-Myc enhancer-driven transcription of aplurality of Myc target genes as described in U.S. Application Ser. No.61/621,897, the entirety of which is hereby incorporated by referenceherein.

In some embodiments, an agent of the present invention is administeredin combination with a cancer therapeutic agent. It should be appreciatedthat the combined administration of an agent of the present inventionand a cancer therapeutic agent can be achieved by formulating the cancertherapeutic agent and agent in the same composition or by administeringthe cancer therapeutic agent and agent separately (e.g., before, after,or interspersed with doses or administration of the cancer therapeuticagent). In some embodiments, an agent of the present invention isadministered to a patient undergoing conventional chemotherapy and/orradiotherapy. In some embodiments the cancer therapeutic agent is achemotherapeutic agent. In some embodiments the cancer therapeutic agentis an immunotherapeutic agent. In some embodiments the cancertherapeutic agent is a radiotherapeutic agent.

Exemplary chemotherapeutic agents that can be administered incombination with the agents of the present invention (e.g., agents thatdisrupt the function of super-enhancers) include alkylating agents (e.g.cisplatin, carboplatin, oxaloplatin, mechlorethamine, cyclophosphamide,chorambucil, nitrosureas); anti-metabolites (e.g. methotrexate,pemetrexed, 6-mercaptopurine, dacarbazine, fludarabine, 5-fluorouracil,arabinosycytosine, capecitabine, gemcitabine, decitabine); plantalkaloids and terpenoids including vinca alkaloids (e.g. vincristine,vinblastine, vinorelbine), podophyllotoxin (e.g. etoposide, teniposide),taxanes (e.g. paclitaxel, docetaxel); topoisomerase inhibitors (e.g.notecan, topotecan, amasacrine, etoposide phosphate); antitumorantibiotics (dactinomycin, doxorubicin, epirubicin, and bleomycin);ribonucleotides reductase inhibitors; antimicrotubules agents; andretinoids. (See, e.g., Cancer: Principles and Practice of Oncology (V.T. DeVita, et al., eds., J.B. Lippincott Company, 9^(th) ed., 2011;Brunton, L, et al. (eds.) Goodman and Gilman's The Pharmacological Basisof Therapeutics, 12^(th) Ed., McGraw Hill, 2010).

Exemplary immunotherapeutic agents include cytokines, such as, forexample interleukin-1 (IL-1), IL-2, IL-4, IL-5, IL-13, IL-7, IL-10,IL-12, IL-15, IL-18, CSF-GM, CSF-G, IFN-γ, IFN-α, TNF, TGF-β but notlimited thereto.

In some embodiments an agent of the present invention can be linked orconjugated to a delivery vehicle, which may also contain cancertherapeutic. Suitable delivery vehicles include liposomes (Hughes et al.Cancer Res 49(22):6214-20, 1989, which is hereby incorporated byreference in its entirety), nanoparticles (Farokhzad et al. Proc Nat'lAcad Sci USA 103(16):6315-20, 2006, which is hereby incorporated byreference in its entirety), biodegradable microspheres, microparticles,and collagen minipellets. The delivery vehicle can contain any of theagents and/or compositions of the present invention, as well aschemotherapeutic, radiotherapeutic, or immunotherapeutic agentsdescribed supra.

In some embodiments an agent of the present invention can be conjugatedto a liposome delivery vehicle (Sofou and Sgouros, Exp Opin Drug Deliv.5(2):189-204, 2008, which is hereby incorporated by reference in itsentirety). Liposomes are vesicles comprised of one or moreconcentrically ordered lipid bilayers which encapsulate an aqueousphase. Suitable liposomal delivery vehicles are apparent to thoseskilled in the art. Different types of liposomes can be preparedaccording to Bangham et al. J. Mol. Biol. 13:238-52, 1965; U.S. Pat. No.5,653,996 to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No.5,885,613 to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau & Kaneda;and U.S. Pat. No. 5,059,421 to Loughrey et al., which are herebyincorporated by reference in their entirety.

These liposomes can be produced such that they contain, in addition tothe therapeutic agents of the present invention, other therapeuticagents, such as immunotherapeutic cytokines, which would then bereleased at the target site (e.g., Wolff et al., Biochim. Biophys. Acta.802:259-73, 1984, which is hereby incorporated by reference in itsentirety).

The present invention also contemplates a composition comprising anagent of the present invention and a pharmaceutically acceptablecarrier, diluent, or excipient. Therapeutic formulations of the agentsof the present invention can be prepared having the desired degree ofpurity with optional pharmaceutically acceptable carriers, excipients orstabilizers (REMINGTON'S PHARMACEUTICAL SCIENCES (A. Osol ed. 1980),which is hereby incorporated by reference in its entirety), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as acetate,Tris-phosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; tonicifiers such as trehaloseand sodium chloride; sugars such as sucrose, mannitol, trehalose orsorbitol; surfactant such as polysorbate; salt-forming counter-ions suchas sodium; metal complexes (e.g., Zn-protein complexes); and/ornon-ionic surfactants such as TWEEN.RTM., PLURONICS.RTM. or polyethyleneglycol (PEG).

The active therapeutic ingredients of the pharmaceutical compositionsalone or in combination with or linked to a cancer therapeutic agent orradiotherapeutic agent) can be entrapped in microcapsules prepared usingcoacervation techniques or by interfacial polymerization, e.g.,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (e.g., liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in REMINGTON'S PHARMACEUTICAL SCIENCES (A. Osol ed. 1980),which is hereby incorporated by reference in its entirety. In someembodiments the agents of the present invention can be conjugated to themicrocapsule delivery vehicle to target the delivery of the therapeuticagent to the site of the cells exhibiting super-enhancer associatedoncogenes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody or polypeptide, whichmatrices are in the form of shaped articles, e.g., films ormicrocapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and.gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT.RTM. (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid.

In some embodiments, an agent of the present invention can be providedwith an enteric coating or otherwise protected from hydrolysis or lowstomach pH. The therapeutically effective compositions containing theagents of the present invention are administered to a subject, inaccordance with known methods, such as intravenous administration, e.g.,as a bolus or by continuous infusion over a period of time, byintramuscular, intraperitoneal, intracerobrospinal, subcutaneous,intra-articular, intrasynovial, intrathecal, oral, topical, orinhalation routes.

Other therapeutic regimens may be combined with the administration ofthe agents of the present invention. The combined administrationincludes co-administration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.Preferably such combined therapy results in a synergistic therapeuticeffect. In some embodiments, a composition of the present invention isadministered in combination with a therapy selected from the groupconsisting of chemotherapy, radiotherapy, proton therapy, surgery, andcombinations thereof.

The composition can include any number of additional active ingredientswhich can act in concert to provide a therapeutic effect, (e.g., asynergistic therapeutic effect), such as a chemotherapeutic agent, aradiotherapeutic agent, a nutritional supplement (e.g. vitamins), anantioxidant, and combinations thereof.

An “effective amount” or “effective dose” of an agent (or compositioncontaining such agent) generally refers to the amount sufficient toachieve a desired biological and/or pharmacological effect, e.g., whencontacted with a cell in vitro or administered to a subject according toa selected administration form, route, and/or schedule. As will beappreciated by those of ordinary skill in the art, the absolute amountof a particular agent or composition that is effective may varydepending on such factors as the desired biological or pharmacologicalendpoint, the agent to be delivered, the target tissue, etc. Those ofordinary skill in the art will further understand that an “effectiveamount” may be contacted with cells or administered in a single dose, orthrough use of multiple doses, in various embodiments. It will beunderstood that agents, compounds, and compositions herein may beemployed in an amount effective to achieve a desired biological and/ortherapeutic effect.

In certain aspects, the present invention relates to a method oftreating a proliferative disorder in a patient in need of suchtreatment, said proliferative disorder characterized by anoncogene-associated super-enhancer occupied by more Mediator or BRD4than an average single enhancer, comprising administering to the patientan effective amount of an agent that disrupts the function of theoncogene-associated super-enhancer, thereby selectively inhibitingproliferation of the oncogene in the patient.

It should be apparent to those skilled in the art that any of thecompounds or agents described above can be employed in the method oftreating the proliferative disorder to achieve the desired result ofdisrupting the function of the super-enhancer. The present inventioncontemplates the treatment of any proliferative disorder (e.g., cancer)that is characterized by an oncogene-associated super-enhancer. In someembodiments, the proliferative disorder to be treated is a hematologicalmalignancy. In some embodiments, the proliferative disorder to betreated is selected from the group consisting of acute lymphoblasticleukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenousleukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma(CTCL), peripheral T-cell lymphoma (PTCL), Mantle cell lymphoma, B-celllymphoma, acute lymphoblastic T cell leukemia (T-ALL), acutepromyelocytic leukemia, and multiple myeloma. In some embodiments, theproliferative disorder is a non-hematological malignancy.

In certain exemplary embodiments, the agent is a BRD4 inhibitor, forexample, small molecule JQ1 or iBET.

In some aspects, the present invention relates to a method of treatingmultiple myeloma involving an IGH-MYC locus that results in aberrantexpression of oncogene c-Myc, comprising administering to a patient inneed of such treatment an effective amount of an agent that decreasesoccupancy levels of BRD4 and MED1 at a super-enhancer associated withthe IGH-MYC locus, wherein decreased occupancy levels of BRD4 and MED1at the super-enhancer disrupt function of the super-enhancer therebydecreasing aberrant expression of oncogene c-Myc such that the multiplemyeloma is treated. In some embodiments, the agent is a BRD4 inhibitor,for example, JQ1 or iBET.

In some aspects, the present invention relates to a method ofidentifying an agent that disrupts a super-enhancer associated with agene, comprising: (a) providing a cell or cell free system comprising asuper-enhancer, or functional fragment and/or variant thereof, and anassociated gene, e.g., a reporter gene; (b) contacting the cell with atest agent, e.g., under conditions suitable for the super-enhancer todrive expression of the associated gene, e.g., to drive expression at apreselected level, e.g., a high level; (c) and measuring the level ofexpression of the associated gene.

In an embodiment decreased expression of the associated gene in thepresence of the test agent indicates that the test agent is as an agentthat disrupts the super-enhancer associated with the gene.

In an embodiment the method comprises transfecting a cell with asuper-enhancer and the associated gene under conditions suitable for thesuper-enhancer to drive high levels of expression of the associatedgene.

In an embodiment the method comprises comparing the level of expressionwith a reference, e.g., expression in a similar system not contactedwith the test agent.

In an embodiment the method comprises confirming disruption of thesuper-enhancer, or functional fragment and/or variant thereof, e.g., byanalysis of the presence of one or more super-enhancer component.

In an embodiment the method is first performed in a cell-free system andrepeated in cell preparation, e.g., a cultured cell.

In an embodiment the method is first performed in a cell-free system ora cell preparation, e.g., a cultured cell, and repeated in an animal.

In an embodiment the super-enhancer is associated with a gene that isexpressed in a disease state cell, e.g., a cancer cell.

In an embodiment the method comprises memorializing the results.

A wide variety of test agents can be used in the methods. For example, atest agent can be a small molecule, polypeptide, peptide, nucleic acid,oligonucleotide, lipid, carbohydrate, or hybrid molecule. Compounds canbe obtained from natural sources or produced synthetically. Compoundscan be at least partially pure or may be present in extracts or othertypes of mixtures. Extracts or fractions thereof can be produced from,e.g., plants, animals, microorganisms, marine organisms, fermentationbroths (e.g., soil, bacterial or fungal fermentation broths), etc. Insome embodiments, a compound collection (“library”) is tested. Thelibrary may comprise, e.g., between 100 and 500,000 compounds, or more.Compounds are often arrayed in multwell plates. They can be dissolved ina solvent (e.g., DMSO) or provided in dry form, e.g., as a powder orsolid. Collections of synthetic, semi-synthetic, and/or naturallyoccurring compounds can be tested. Compound libraries can comprisestructurally related, structurally diverse, or structurally unrelatedcompounds. Compounds may be artificial (having a structure invented byman and not found in nature) or naturally occurring. In some embodimentsa library comprises at least some compounds that have been identified as“hits” or “leads” in other drug discovery programs and/or derivativesthereof. A compound library can comprise natural products and/orcompounds generated using non-directed or directed synthetic organicchemistry. Often a compound library is a small molecule library. Otherlibraries of interest include peptide or peptoid libraries, cDNAlibraries, and oligonucleotide libraries. A library can be focused(e.g., composed primarily of compounds having the same core structure,derived from the same precursor, or having at least one biochemicalactivity in common).

Compound libraries are available from a number of commercial vendorssuch as Tocris BioScience, Nanosyn, BioFocus, and from governmententities. For example, the Molecular Libraries Small Molecule Repository(MLSMR), component of the U.S. National Institutes of Health (NIH)Molecular Libraries Program is designed to identify, acquire, maintain,and distribute a collection of >300,000 chemically diverse compoundswith known and unknown biological activities for use, e.g., inhigh-throughput screening (HTS) assays (see https://mli.nih.gov/mli/).The NIH Clinical Collection (NCC) is a plated array of approximately 450small molecules that have a history of use in human clinical trials.These compounds are highly drug-like with known safety profiles. The NCCcollection is arrayed in six 96-well plates. 50 μl of each compound issupplied, as an approximately 10 mM solution in 100% DMSO. In someembodiments, a collection of compounds comprising “approved human drugs”is tested. An “approved human drug” is a compound that has been approvedfor use in treating humans by a government regulatory agency such as theUS Food and Drug Administration, European Medicines Evaluation Agency,or a similar agency responsible for evaluating at least the safety oftherapeutic agents prior to allowing them to be marketed. The test agentmay be, e.g., an antineoplastic, antibacterial, antiviral, antifungal,antiprotozoal, antiparasitic, antidepressant, antipsychotic, anesthetic,antianginal, antihypertensive, antiarrhythmic, antiinflammatory,analgesic, antithrombotic, antiemetic, immunomodulator, antidiabetic,lipid- or cholesterol-lowering (e.g., statin), anticonvulsant,anticoagulant, antianxiety, hypnotic (sleep-inducing), hormonal, oranti-hormonal drug, etc. In some embodiments, a compound is one that hasundergone at least some preclinical or clinical development or has beendetermined or predicted to have “drug-like” properties. For example, thetest agent may have completed a Phase I trial or at least a preclinicalstudy in non-human animals and shown evidence of safety andtolerability. In some embodiments, a test agent is substantiallynon-toxic to cells of an organism to which the compound may beadministered or cells in which the compound may be tested, at theconcentration to be used or, in some embodiments, at concentrations upto 10-fold, 100-fold, or 1,000-fold higher than the concentration to beused. For example, there may be no statistically significant adverseeffect on cell viability and/or proliferation, or the reduction inviability or proliferation can be no more than 1%, 5%, or 10% in variousembodiments.

In various embodiments of any aspect herein pertaining to screeningmethods (e.g., methods of identifying agents), the screen may beperformed using a single test agent or multiple test agents in a givenreaction vessel. In various embodiments the number of reaction vesselsand/or test agents is at least 10; 100; 1000; 10,000; 100,000, or more.In some embodiments of any aspect herein pertaining at least in part toscreening methods (e.g., methods of identifying agents) a highthroughput screen (HTS) is performed. High throughput screens ofteninvolve testing large numbers of test agents with high efficiency, e.g.,in parallel. For example, tens or hundreds of thousands of agents may beroutinely screened in short periods of time, e.g., hours to days. Suchscreening is often performed in multiwell plates (sometimes referred toas microwell or microtiter plates or microplates) containing, e.g., 96,384, 1536, 3456, or more wells or other vessels in which multiplephysically separated depressions, wells, cavities, or areas(collectively “wells”) are present in or on a substrate. Different testagent(s) may be present in or added to the different wells. It will beunderstood that some wells may be empty, may comprise replicates, or maycontain control agents or vehicle. High throughput screens may involveuse of automation, e.g., for liquid handling, imaging, and/or dataacquisition or processing, etc. In some embodiments an integrated robotsystem comprising one or more robots transports assay-microplates fromstation to station for, e.g., addition, mixing, and/or incubation ofassay constituents (e.g., test agent, target, substrate) and, in someembodiments, readout or detection. A HTS system may prepare, incubate,and analyze many plates simultaneously. Certain general principles andtechniques that may be applied in embodiments of a HTS are described inMacarrón R & Hertzberg R P. Design and implementation of high-throughputscreening assays. Methods Mol Biol., 565:1-32, 2009 and/or An W F &Tolliday N J., Introduction: cell-based assays for high-throughputscreening. Methods Mol Biol. 486:1-12, 2009, and/or references in eitherof these. Exemplary methods are also disclosed in High ThroughputScreening: Methods and Protocols (Methods in Molecular Biology) byWilliam P. Janzen (2002) and High-Throughput Screening in Drug Discovery(Methods and Principles in Medicinal Chemistry) (2006) by Jorg Hüser.Test agent(s) showing an activity of interest (sometimes termed “hits”)may be retested and/or, optionally (e.g., depending at least in part onresults of retesting) selected for further testing, development, or use.In some embodiments one or more structural analogs of a hit issynthesized. Such analogs may, for example, comprise substitution of oneor more functional groups or heteroatoms present in the hit by adifferent functional group or heteroatom or substituting a heteroatom orfunctional group present in place of a hydrogen in the hit, etc. In someembodiments one or more such analog(s) are then tested for a property oractivity of interest (e.g., ability to disrupt a super-enhancerassociated with an oncogene or disease related gene).

Positive and/or negative controls may be used in any of the screens. Anappropriate positive or negative control can be selected based at leastin part on the assay. A negative control may be to perform the assay inthe absence of a test agent.

In some embodiments, information derived from sequence analysis,mutational analysis, and/or structural analysis is used in theidentification of a modulator, e.g., an agent that interferes withtranscriptional coactivator or BRD4 co-occupancy of super-enhancers andactive transcription start sites. For example, in some embodiments astructure (e.g., a two-dimensional or three-dimensional structure) of atarget, e.g., a TF, generated at least in part using, e.g., nuclearmagnetic resonance, homology modeling, and/or X-ray crystallography isused. In some embodiments a structure obtained with a ligand (e.g., aninhibitor) bound to the target may be used. In some embodiments acomputer-aided computational approach sometimes referred to as “virtualscreening” is used in the identification of candidate modulators,Structures of compounds, e.g., small molecules may be screened forability to bind to a region (e.g., a “pocket”) accessible to thecompound. The region may be any region accessible to the compound, e.g.,a concave region on the surface or a cleft or a region involved indimerization. A variety of docking and pharmacophore-based algorithmsare known in the art, and computer programs implementing such algorithmsare available. Commonly used programs include Gold, Dock, Glide, FlexX,Fred, and LigandFit (including the most recent releases thereof). See,e.g., Ghosh, S., et al., Current Opinion in Chemical Biology, 10(3):194-2-2, 2006; McInnes C., Current Opinion in Chemical Biology; 11(5):494-502, 2007, and references in either of the foregoing articles, whichare incorporated herein by reference. In some embodiments a virtualscreening algorithm may involve two major phases: searching (also called“docking”) and scoring. During the first phase, the programautomatically generates a set of candidate complexes of two molecules(test compound and target molecule) and determines the energy ofinteraction of the candidate complexes. The scoring phase assigns scoresto the candidate complexes and selects a structure that displaysfavorable interactions based at least in part on the energy. To performvirtual screening, this process may be repeated with a large number oftest compounds to identify those that, for example, display the mostfavorable interactions with the target. In some embodiments, low-energybinding modes of a small molecule within an active site or possibleactive site or other target region are identified. In some embodiments acompound capable of docking at a site where mutations are known toinhibit activity of the target is identified. Variations may include theuse of rigid or flexible docking algorithms and/or including thepotential binding of water molecules. In some embodiments thethree-dimensional structure of an enzyme's active site may be used toidentify potential inhibitors. Agent(s) that have the potential to bindin or near an active site may be identified. These predictions may thenbe tested using the actual compound. A new inhibitor thus identified maythen be used to obtain a structure of the enzyme in an inhibitor/enzymecomplex to show how the molecule is binding to the active site. Furtherchanges may be made to the inhibitor, e.g., to try to improve binding.This cycle may be repeated until an inhibitor of sufficient predicted oractual potency (e.g., a desired potency for therapeutic purposes) isidentified. Numerous small molecule structures are available and can beused for virtual screening. A collection of compound structures maysometimes referred to as a “virtual library”. For example, ZINC is apublicly available database containing structures of millions ofcommercially available compounds that can be used for virtual screening(http://zinc.docking.org/; Shoichet, J. Chem. Inf. Model., 45(1):177-82,2005). A database containing about 250,000 small molecule structures isavailable on the National Cancer Institute (U.S.) website (athttp://129.43.27.140/ncidb2/). In some embodiments multiple smallmolecules may be screened, e.g., up to 50,000; 100,000; 250,000;500,000, or up to 1 million, 2 million, 5 million, 10 million, or more.Compounds can be scored and, optionally, ranked by their potential tobind to a target. Compounds identified in virtual screens can be testedin cell-free or cell-based assays or in animal models to confirm theirability to inhibit activity of a target molecule, their ability toactivate a target molecule, and/or to assess their biological and/orpharmacological activity. Computational approaches may be used topredict one or more physico-chemical, pharmacokinetic and/orpharmacodynamic properties of compounds identified in a physical orvirtual screen. Such information may be used, e.g., to select one ormore hits for, e.g., further testing, development, or use. For example,small molecules having characteristics typical of “drug-like” moleculesmay be selected and/or small molecules having one or more undesiredcharacteristics may be avoided.

In some aspects of any screening and/or characterization methods, testagents are contacted with test cells (and optionally control cells) orused in cell-free assays at a predetermined concentration. In someembodiment the concentration is about up to 1 nM. In some embodimentsthe concentration is between about 1 nM and about 100 nM. In someembodiments the concentration is between about 100 nM and about 10 μM.In some embodiments the concentration is at or above 10 μM, e.g.,between 10 μM and 100 μM. Following incubation for an appropriate time,optionally a predetermined time, the effect of compounds or compositionon a parameter of interest in the test cells is determined by anappropriate method known to one of ordinary skill in the art, e.g., asdescribed herein. Cells can be contacted with compounds for variousperiods of time. In certain embodiments cells are contacted for between12 hours and 20 days, e.g., for between 1 and 10 days, for between 2 and5 days, or any intervening range or particular value. Cells can becontacted transiently or continuously. If desired, the compound can beremoved prior to assessing the effect on the cells.

One skilled in the art readily appreciates that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The details of thedescription and the examples herein are representative of certainembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Modifications therein and other uses will occurto those skilled in the art. These modifications are encompassed withinthe spirit of the invention. It will be readily apparent to a personskilled in the art that varying substitutions and modifications may bemade to the invention disclosed herein without departing from the scopeand spirit of the invention.

The articles “a” and “an” as used herein in the specification and in theclaims, unless clearly indicated to the contrary, should be understoodto include the plural referents. Claims or descriptions that include“or” between one or more members of a group are considered satisfied ifone, more than one, or all of the group members are present in, employedin, or otherwise relevant to a given product or process unless indicatedto the contrary or otherwise evident from the context. The inventionincludes embodiments in which exactly one member of the group is presentin, employed in, or otherwise relevant to a given product or process.The invention also includes embodiments in which more than one, or allof the group members are present in, employed in, or otherwise relevantto a given product or process. Furthermore, it is to be understood thatthe invention provides all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim dependent on the same base claim (or, as relevant, any otherclaim) unless otherwise indicated or unless it would be evident to oneof ordinary skill in the art that a contradiction or inconsistency wouldarise. It is contemplated that all embodiments described herein areapplicable to all different aspects of the invention where appropriate.It is also contemplated that any of the embodiments or aspects can befreely combined with one or more other such embodiments or aspectswhenever appropriate. Where elements are presented as lists, e.g., inMarkush group or similar format, it is to be understood that eachsubgroup of the elements is also disclosed, and any element(s) can beremoved from the group. It should be understood that, in general, wherethe invention, or aspects of the invention, is/are referred to ascomprising particular elements, features, etc., certain embodiments ofthe invention or aspects of the invention consist, or consistessentially of, such elements, features, etc. For purposes of simplicitythose embodiments have not in every case been specifically set forth inso many words herein. It should also be understood that any embodimentor aspect of the invention can be explicitly excluded from the claims,regardless of whether the specific exclusion is recited in thespecification. For example, any one or more nucleic acids, polypeptides,cells, species or types of organism, disorders, subjects, orcombinations thereof, can be excluded.

Where the claims or description relate to a composition of matter, e.g.,a nucleic acid, polypeptide, cell, or non-human transgenic animal, it isto be understood that methods of making or using the composition ofmatter according to any of the methods disclosed herein, and methods ofusing the composition of matter for any of the purposes disclosed hereinare aspects of the invention, unless otherwise indicated or unless itwould be evident to one of ordinary skill in the art that acontradiction or inconsistency would arise. Where the claims ordescription relate to a method, e.g., it is to be understood thatmethods of making compositions useful for performing the method, andproducts produced according to the method, are aspects of the invention,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where ranges are given herein, the invention includes embodiments inwhich the endpoints are included, embodiments in which both endpointsare excluded, and embodiments in which one endpoint is included and theother is excluded. It should be assumed that both endpoints are includedunless indicated otherwise. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of ordinary skill in the art, values that areexpressed as ranges can assume any specific value or subrange within thestated ranges in different embodiments of the invention, to the tenth ofthe unit of the lower limit of the range, unless the context clearlydictates otherwise. It is also understood that where a series ofnumerical values is stated herein, the invention includes embodimentsthat relate analogously to any intervening value or range defined by anytwo values in the series, and that the lowest value may be taken as aminimum and the greatest value may be taken as a maximum. Numericalvalues, as used herein, include values expressed as percentages. For anyembodiment of the invention in which a numerical value is prefaced by“about” or “approximately”, the invention includes an embodiment inwhich the exact value is recited. For any embodiment of the invention inwhich a numerical value is not prefaced by “about” or “approximately”,the invention includes an embodiment in which the value is prefaced by“about” or “approximately”. “Approximately” or “about” generallyincludes numbers that fall within a range of 1% or in some embodimentswithin a range of 5% of a number or in some embodiments within a rangeof 10% of a number in either direction (greater than or less than thenumber) unless otherwise stated or otherwise evident from the context(except where such number would impermissibly exceed 100% of a possiblevalue). It should be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. It should also beunderstood that unless otherwise indicated or evident from the context,any product or composition described herein may be considered“isolated”.

EXAMPLES Example 1 Master Transcription Factors and Mediator EstablishSuper-Enhancers at Key Cell Identity Genes Introduction

Transcription factors typically regulate gene expression by bindingcis-acting regulatory elements known as enhancers and recruitingcoactivators and RNA Polymerase II (RNA Pol II) to target genes (Ong andCorces, 2011). Transcription factor-bound enhancers interact with targetgene promoters via DNA looping events facilitated by the Mediatorco-activator complex and cohesin (Kagey et al., 2010). Between 400,000and 1.4 million putative enhancers have been identified in the mammaliangenome (Bernstein et al., 2012; Thurman et al., 2012). In any one celltype, the number of active enhancers is estimated to be in the thousandsand enhancer activity is largely cell-type specific (Bernstein et al.,2012; Shen et al., 2012; Yip et al., 2012). Whereas most genes aretranscriptionally active in multiple cell types, enhancers tend to beactive only in specific lineages (Shen et al., 2012). These data suggestthat much of the transcriptional control of mammalian development is dueto the diverse activity of enhancers that control cell type specificpatterns of gene expression.

In embryonic stem cells (ESCs), control of the gene expression programthat establishes and maintains ESC state is dependent on a remarkablysmall number of master transcription factors (Young, 2011). Thesetranscription factors, which include Oct4, Sox2 and Nanog (OSN), bind toapproximately 7,000 enhancers together with the Mediator coactivatorcomplex (Kagey et al., 2010). The Mediator complex facilitates theability of enhancer-bound transcription factors to recruit RNA Pol II tothe promoters of target genes (Malik and Roeder, 2010) and is essentialfor maintenance of ESC state and early embryonic development (Kagey etal., 2010). Reduced levels of either Oct4 or Mediator have a verysimilar effect on the ESC gene expression program and cause the samerapid loss of ESC identity (Kagey et al., 2010).

It is striking that ESC maintenance is highly sensitive to perturbationsin the levels of Mediator (Kagey et al., 2010). To understand thereasons underlying this hypersensitivity, we investigated, enhancersbound by Mediator in these cells. We identified approximately 200genomic regions that contained tightly spaced clusters of enhancersspanning extraordinarily large domains. These “super-enhancers” wereoccupied by an order of magnitude more Mediator than the averageenhancer, and were associated with the key cell-type specific ESC genes.These enhancers also conferred stronger enhancer activity relative tothe average enhancer, suggesting these elements drive gene expressionprograms and cell state. During ESC differentiation, the ESCsuper-enhancers were rapidly lost and new super-enhancers were formed atgenes key to the differentiated cell type. Additional cell types werefound to have super-enhancers associated with highly expressed andcell-type specific genes. These results argue that super-enhancers drivegenes essential for cell identity in multiple cell types and that theseelements are especially sensitive to perturbations involved in dynamicchanges in cell state.

Results Large Genomic Domains Occupied by Mediator in ESCs

Previous studies have shown that co-occupancy of sites by the Oct4, Sox2and Nanog transcription factors is highly predictive of enhanceractivity (Chen et al., 2008). We generated ChIP-Seq data for Oct4, Sox2,Nanog (OSN) in murine ESCs and identified 6,343 regions that were boundby all three transcription factors. The Mediator co-activator complexhas been previously shown to interact with the enhancer-boundtranscription factors and facilitate recruitment of the transcriptionapparatus to active gene promoters (Malik and Roeder, 2010). Analysis ofthe 6,343 OSN regions confirmed the presence of Mediator, includingregions surrounding the Klf4 gene (FIG. 1A). Therefore, we defined the6,343 regions bound by OSN as ESC enhancers.

Closer inspection of the 6,343 ESC enhancers revealed a surprisingfeature: some ESC enhancers are occupied by extremely high levels ofMediator (FIG. 1B). Global analysis of the 6,343 ESC enhancers confirmedthe distribution of Mediator occupancy across this set of regions is notevenly distributed (FIG. 1C). Instead, there is a distribution ofoccupancy that indicates these regions fall into two distinct classes,with one class containing an exceptional amount of Mediator proteins(FIG. 1C). Further analysis of this small subset (211) of regionsrevealed that, on average, they contained 27 times more Mediatorproteins compared to the remaining 6,132 enhancers (FIG. 1D).Additionally, on average these regions covered larger genomic distances(5.2 kb) compared to the remaining enhancers (469 bp) (FIG. 1D). Thus,these ˜200 regions, which we call “super-enhancers”, are occupied by atleast an order of magnitude more Mediator relative to the mean, andtypically span DNA domains at least an order of magnitude larger.

Many genome wide enhancer mapping efforts utilize histone marks andregulatory proteins as surrogates for enhancers (Bernstein et al., 2012;Shen et al., 2012). Further characterization of the super-enhancersrevealed that these regions are also occupied by otherenhancer-associated modifications and proteins, including H3K27ac, ahistone modification commonly found at enhancers and used to predictregions of enhancers activity (Creyghton et al., 2010; Rada-Iglesias etal., 2011). Interestingly, H3K27Ac failed to reveal the strikingdisparity noted for OSN-Mediator bound super-enhancers. Thus, MediatorChIP-Seq data is superior to surrogate data from histone modificationsfor identifying super-enhancers in ESCs.

Super-Enhancers are Associated with Key ESC Genes

Most studies have assigned enhancers to putative target genes by usingthe proximity of enhancers and target genes. Recent work has identifiedtopological domains associated with transcriptional control in the ESCgenome using high throughput chromatin conformation capture data(Hi-C)(Dixon et al., 2012). We therefore used proximity of enhancerelements and genes to facilitate mapping of ESC enhancers to promoters,and further used Hi-C to additionally assign enhancers to promoters ofgenes that were greater than 40 kb away. Previous studies usingchromatin configuration capture (3C) have shown that, at an enhancerelement brought into close proximity to a promoter region by DNAlooping, the Mediator ChIP-Seq signals are similar at both regions(Kagey et al., 2010). We therefore required that enhancer-promoterinteraction candidates have similar levels of Mediator. The assignmentsof super-enhancers to promoters identified 192 genes, with a further˜5,300 assigned by Hi-C. For three of these genes, the proximity betweenportions of the super-enhancer and the target promoter were previouslyestablished using 3C (Kagey et al., 2010).

A global RNA sequencing (RNA-Seq) analysis of the genes assigned to ESCenhancers confirmed that these genes were expressed at very high levelscompared to other genes in ESCs (FIG. 2A). Further examination of thisset of genes, however, revealed a striking difference: thesuper-enhancer-associated genes were expressed at higher levels comparedto those neighboring the remaining enhancers (FIG. 2B,C), Compared tothe average expression levels of genes near the median enhancer (1.84RPKM), genes associated with super-enhancers were expressed 6-timeshigher (FIG. 2A). These results suggest super-enhancers are associatedwith the most highly expressed genes compared to other enhancers.

We next determined if these highly expressed genes were important forESC identity. In contrast to the other highly expressed genes that werefound near the 6,132 enhancers, including house-keeping genes,super-enhancer-associated genes are critical for ESC maintenance andreprogramming. Super-enhancers were directly associated with many genespreviously shown to play important roles in ESC identity, includingEsrrb (Ivanova et al., 2006; Zhang et al., 2008); Tbx3 (Ivanova et al.,2006; Niwa et al., 2009); and the mir290-295 microRNA gene cluster(Lichner et al., 2011; Marson et al., 2008; Zovoilis et al., 2009).Remarkably, the super-enhancer-associated genes included those encodingthe ESC master transcription factors Oct4, Sox2 and Nanog (FIG. 2D).These three transcription factors are known to auto-regulate theirexpression through promoter binding, forming an interconnectedauto-regulatory loop. This form of auto-regulation is a core feature ofthe ESC transcriptional regulatory circuitry (Boyer et al., 2005), whoseestablishment is likely key to reprogramming of various cells into iPScells (Jaenisch and Young, 2008). Small portions of the super-enhancersassociated with these genes have previously been shown to have enhanceractivity in reporter assays (Chen et al., 2008) and to participate inenhancer-promoter looping at the Oct4 and Nanog genes (Kagey et al.,2010). Thus, the genes encoding the master transcription factors arethemselves under the control of super-enhancers. Overall these resultssupport a model that super-enhancers associate with highly expressed andhighly cell-type specific genes that include key drivers of ESCidentity.

Super-Enhancers Confer Strong Enhancer Activity

One striking feature of the super-enhancers is that they containmultiple, highly enriched regions of Mediator compared to averageenhancers that typically consist of a single peak of the coactivator(FIG. 3A). DNA sequence analysis confirmed that super-enhancerscontained more OSN binding motifs than do median enhancers (FIG. 3A). Totest whether these super-enhancers confer stronger enhancer activitythan median enhancers, we cloned 3 kb regions of super-enhancers andmedian enhancers into luciferase reporter constructs that weresubsequently transfected into ESCs. We found that on average,super-enhancers drove 16 times more luciferase expression than medianenhancers (FIG. 3B). Since a super-enhancer contained more Mediatoroccupancy compared to a median enhancer, these results suggested thatclusters of enhancers may display higher enhancer activity in ESCs. Totest this model, we generated an artificial super-enhancer byoligomerizing the distal median enhancer of the Sgk1 gene. As a singlemedian enhancer, this region displayed low luciferase activity (FIG.3B). Remarkably, the dimeric Sgk1 enhancer exhibited 2-times higheractivity, while the tetrameric Sgk1 enhancer exhibited 3-times higherenhancer activity compared to the single Sgk1 enhancer drivingluciferase expression in ESCs (FIG. 3C). These results suggest thatsuper-enhancers can be formed by clusters of enhancers, that they havehigher activity than median enhancers, and are sufficient to drive highexpression of key, cell type-specific genes required to maintain ESCidentity.

Rapid Loss of ESC Super-Enhancers During ESC Differentiation

If super-enhancers play key roles in transcriptional control of cellidentity, then differentiation of ESCs should lead to loss of ESCsuper-enhancers. To test this notion, we stimulated ESCs todifferentiate into a trophectoderm lineage by shutting down Oct4transcription (FIG. 4A)(Niwa et al., 2000). Loss of Oct4 results incellular differentiation, loss of expression of Oct4 target genes, andupregulation of the trophectoderm master regulator transcription factorCdx2 (Deb et al., 2006; Niwa et al., 2005; Strumpf et al., 2005; Wang etal., 2010).

The fate of ESC super-enhancers during differentiation was examined byprofiling global levels of Mediator using ChIP-PCR (FIG. 4B). All sevenof the super-enhancers tested that were occupied by OSN and Mediator inESCs had at least two-fold lower levels of Mediator proteins upondifferentiation (FIG. 4B). On average, the tested super-enhancers had68% lower levels of Mediator upon ESC differentiation compared tocontrol ESCs (FIG. 4B). This included the super-enhancers to the key ESCgenes Oct4/Pou5f1 and Sox2 (FIG. 4B). In contrast, four of the fivetypical enhancers that were tested retained high levels of Mediatorcompared to super-enhancers upon ESC differentiation (FIG. 4B).Surprisingly, only one of the typical enhancers tested had at leasttwo-fold lower levels of Mediator upon differentiation (FIG. 4B). Onaverage, median enhancers had only 14% lower levels of Mediator upondifferentiation compared to control ESCs (FIG. 4B). Together, theseresults are consistent with the model that super-enhancers play keyroles in establishing and maintaining cell state, and that theseenhancer elements are sensitive to perturbations that accompany thedynamic changes in cell state during differentiation.

Super-Enhancers are Found in Multiple Cell Types and are Cell-TypeSpecific

The identification of both ESC and trophectoderm lineage super-enhancerssuggest that super-enhancers may be a common feature of mammalian cells.Accordingly, in any given cell type, super-enhancer associated genes arelikely to play prominent roles in establishing and maintaining cellidentity. Further, the pattern of super-enhancers in any given cell typeis likely to be cell-type specific.

To test these predictions, we profiled Mediator levels and mastertranscription factor Pu.1 in pro-B cells using ChIP-Seq. Mediatoroccupancy highly correlated with occupancy of Pu.1 at promoter distalsites (FIG. 5A, B). Of the 13,303 sites bound by Pu.1 in pro-B cells,79% were co-occupied by Mediator. Using similar criteria as in ESCs, 392super-enhancers were identified in pro-B cells, and exhibited extremelyhigh levels of Mediator occupancy (FIG. 5B,C), On average, the pro-Bsuper-enhancers contained 31 times more Mediator proteins compared tothe remaining 12,911 enhancers, and covered larger genomic distances(15.4 kb) compared to the remaining enhancers (422 bp). These findingssupport the conclusion that super-enhancers are a general feature ofmammalian cells.

Genes associated with super-enhancers in pro-B cells were previouslyshown to be important for pro-B cell development, supporting the modelthat super-enhancers drive expression of target genes critical forcellular identity. Among the 355 super-enhancer-associated genes thatare highly expressed in pro-B cells included many genes previously shownto play important roles in B cell development, including Pax5; Rag2;VpreB1 and VpreB2. We next determined if super-enhancers and theirassociated genes are cell-type specific by comparing ESC and pro-B cellsuper-enhancers and their target genes (FIG. 5D,E). The set ofsuper-enhancers showed minimal overlap between ESCs and pro-B cells(FIG. 5D), Of the 211 ESC super-enhancers, only 9 regions (2%)overlapped with the pro-B cell super-enhancers (FIG. 5D). Furthermore,the super-enhancer-associated genes exhibited highly cell-type specificpatterns of expression (FIG. 5D). Of the 192 genes neighboringsuper-enhancers in ESCs, only 15 (8%) were associated withsuper-enhancers in pro-B cells (FIG. 5E). These results suggest thatsuper-enhancers are likely to be a general feature of most cell typesand are likely to drive the expression of genes controlling cellularidentity.

Discussion

We have identified in multiple cell types the existence ofsuper-enhancers. Super-enhancers are enhancers bound by master regulatortranscription factors that contain disproportionately high levels of theMediator co-activator complex. Mediator levels are likely to be ratelimiting for enhancer mediated transcription and as such, the disparityin Mediator levels at super-enhancers potentially represents animportant hierarchical stratification of enhancers. Indeed, in multiplecell types, super-enhancers associate with known genes essential forcell identity and globally are likely to be the drivers of key cellidentity controlling genes.

The observation of super-enhancers also suggests the complexity ofcis-regulating elements can be significantly reduced. Although somewherebetween hundreds of thousand and millions of enhancers are likely toexist in the mammalian genome, in any given cell type only a few hundredsuper-enhancers are likely to drive the expression of genes thatestablish cellular identity. In many cell types, small subsets oftranscriptionally active genes have been identified through geneticscreens as essential for cellular identity. However an analogousappreciation does not exist for enhancers in any given cell types. Thecharacteristic features of super-enhancers strongly suggest that theymay be among the most essential enhancers in any given cell type.

Lastly, the ability of super-enhancers to drive expression of key cellidentity genes suggest that mutations to super-enhancers may potentiallylead to disease and developmental defect. Indeed, recent evidence fromthe ENCODE consortium revealed that the majority of disease associatedSNPs occur in regulatory regions (Bernstein et al., 2012; Schaub et al.,2012). It is easy to imagine that loss of a super-enhancer throughgenetic deletions could lead to developmental defects through theinability to fully establish cellular identity. Conversely,translocation of a super-enhancer could result in aberrant generegulation. Example 2 below provides evidence that super-enhancersassociate with key cancer dependency genes, including c-Myc via thetranslocated IgH super-enhancer in Multiple Myeloma.

The association of super-enhancers with key cell identity genes as wellas cancer dependency genes argues that super-enhancers are important andessential components of cellular identity. Given super-enhancers reflectthe occupancy of master regulator transcription factors in a given celltype, identification of super-enhancers in any cell type couldpotentially facilitate the mapping of the core transcriptionalcircuitry. In disease cells, super-enhancers have the potential to actas powerful biomarkers, identifiers of drug target candidates, and canpotentially they themselves be drugged via targeting of Mediator andother enhancer bound components. More importantly, the characterizationof super-enhancers implores a departure from a gene centric view of thegenome, and instead supports an appreciation that regulatory controlregions found in intergenic DNA may represent key features in theblueprints of mammalian development and disease.

References

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Example 2 Selective Inhibition of Tumor Oncogenes by Disruption ofSuper-Enhancers Introduction

Inhibitors of chromatin regulators are gaining interest as therapeuticagents for cancer because of their ability to specifically repress keyoncogenic drivers in many tumor types. A major challenge in cancertherapeutics has been the direct pharmacologic inhibition of oncogenictranscription factors such as c-MYC. MYC is one of the most commonlyamplified oncogenes in cancer, but lacks clear ligand-binding domains,rendering it difficult to target by small molecule inhibitors (Nair andBurley, 2003). However, several recent studies have shown thatinhibition of chromatin regulators, such as the bromodomain proteinBRD4, may represent an alternate avenue for selectively targeting thesekey oncogenic drivers. It is not yet known how inhibition of a generaltranscriptional regulator can exert a specific effect on a small numberof genes. Understanding this concept will aid the development andselection of drugs in treating many cancers.

BRD4 was first identified as an interaction partner of the murineMediator coactivator complex, and has subsequently been shown toassociate with this transcription complex in a variety of human cells(Dawson et al., 2011; Jiang et al., 1998; Wu et al., 2003). BRD4 is alsoinvolved in the control of transcriptional elongation through itsassociation with the positive transcription elongation factor, P-TEFb(Jang et al., 2005; Yang et al., 2005). In addition, bromodomainproteins can associate with specific acetylated histone residues, aninteraction which can be disrupted by small molecules that competitivelyoccupy the acetyl-lysing binding pockets in select members of this61-member protein family (Filippakopoulos et al., 2012). Two recentlydeveloped bromodomain inhibitors, JQ1 and iBET, selectively bind to BRD4(Filippakopoulos et al., 2010; Nicodeme et al., 2010). Despite thisgeneral role played in transcription regulation, inhibition of BRD4 byBET-inhibitors appears to have a highly selective effect on tumor cells(Dawson et al., 2011; Delmore et al., 2011; Mertz et al., 2011; Zuber etal., 2011). BET-inhibitors appear to cause dramatic suppression of thepotent oncogene, MYC, and lead to a pronounced anti-proliferative effectin a range of tumors, including multiple myeloma (MM), Burkitt'slymphoma (BL), and acute myeloid leukemia (AML) (Dawson et al., 2011;Delmore et al., 2011; Mertz et al., 2011; Zuber et al., 2011). AlthoughBRD4 inhibition shows great promise as a therapeutic agent in cancer, itremains unclear why inhibition of this general chromatin regulator has aselective effect on the MYC gene in these tumor cells.

To investigate this mechanism, we turned to concepts described Example 1above. In that study, we demonstrated that transcriptional activators,such as the Mediator coactivator complex are not distributed evenlythroughout the genome. Instead, we found that Mediator binding isconcentrated at a discrete number of enhancer regions, which we haveclassified as super-enhancers. Our analysis of mouse embryonic stemcells (mESCs) revealed that these “super-enhancers” consist of enhancerclusters that span vast chromatin domains when compared to typicalenhancer regions and are occupied by an order of magnitude more Mediatorcomplex proteins. In addition, super-enhancers preferentially associatewith and activate genes key to cell state.

Enhancers function through co-operative and synergistic interactionsbetween multiple transcription factors and coactivators (Carey, 1998;Carey et al., 1990; Giese et al., 1995; Kim and Maniatis, 1997; Thanosand Maniatis, 1995). Cooperative binding and synergistic activationconfer increased sensitivity, so that small changes in activatorconcentration can lead to dramatic changes in activator binding andtranscription of associated genes (Carey, 1998). This led us tohypothesize that highly sensitive super-enhancers driving key oncogenicdrivers in multiple myeloma may account for the selective effect of BRD4inhibition.

In this study, we show that BRD4 inhibition has a highly selectiveeffect on critical tumor genes associated with super-enhancers. Asexpected, given its role as a general regulator of transcriptional pauserelease and its association with the Mediator complex, we found thatBRD4 was located at a majority of active enhancers and promoters intumor cells. Strikingly, extreme levels of BRD4 were found at a smallsubset of enhancer regions, which we have termed super-enhancers. Theseregions are similar to the super-enhancers described in mouse embryonicstem cells as discussed in Example 1 above. We found that binding ofBRD4 and Mediator at super-enhancers was hyper-sensitive to loss of BRD4binding through BET inhibition. This in turn corresponded to a dramaticloss of transcription at super-enhancer associated genes, such as MYC.Our data suggest a model of how inhibitors of generally acting chromatinregulators can exert a gene-specific effect, through the disruption ofheavily occupied, cooperatively bound sites functioning at highlyexpressed tumor regulators. This concept may improve our understandingof how these drugs should be selected for the treatment ofgenetically-defined cancers.

Results

Mediator and BRD4 Co-Occupy Promoters of Active Genes in MultipleMyeloma

In Example 1 above it was shown that Mediator and BRD4 co-occupyenhancers and active transcription start sites in embryonic stem cellsand in differentiated cells. To determine whether Mediator and BRD4co-occupy these sites in multiple myeloma cells, we used chromatinimmunoprecipitation coupled to high-throughput sequencing (ChIP-Seq)with antibodies directed against Mediator, Brd4 and various marks ofenhancers and active transcription start sites in MM.1S cells (FIG. 6).The results, whether viewed by individual genes tracks or by meta-geneanalysis, show that Mediator and BRD4 generally co-occupy enhancers andactive transcription start sites (FIG. 6A, B). Signals for Mediator andBRD4 were found together with those for nucleosomes with the histonemodification H3K27Ac in 8,000 regions lacking transcription start sites,and these were considered enhancers. Signals for BRD4 and Mediator werealso found together with those for the histone modification H3K4me3 andRNA polymerase II at 14,000 annotated transcription start sites, andthese were considered active transcription start sites. The levels ofMediator and BRD4 occupancy correlated with one another at bothenhancers and transcription start sites (FIG. 6C), and the levels ofBRD4 were correlated with the levels of RNA polymerase II at genes (FIG.6D), consistent with the results observed in non-tumor cells in Example1 above. These results indicate that Mediator and BRD4 generallyco-occupy enhancers and active transcription start sites throughout thegenome of MM.1S cells.

Super-Enhancers are Associated with Key Multiple Myeloma Genes

The sizes of enhancers identified by Mediator occupancy showed anunusual distribution, with a small subset of enhancers containingexceptional levels of Mediator protein (FIG. 7A). These 210“super-enhancers” have features similar to those described in Example 1above for mESCs (FIG. 7A). These are regions occupied, on average, by16-fold more Mediator compared to normal enhancer regions.Super-enhancers also occupy larger genomic regions than normalenhancers, with a median size of 20 kb, 16-fold greater than the normalenhancer size of 1.3 kb. In addition to high Mediator occupancy, theseenhancers were also bound by exceptional levels of BRD4, on average,16-fold higher than normal enhancers (FIG. 7B).

As noted in Example 1 above, in ESCs and in differentiated cells,super-enhancers have exceptional transcription activation activity andare associated with highly expressed cell-type-specific genes that arelocated nearby. In MM.1S cells, super-enhancers were associated withhighly expressed, cell-type specific genes, including genes known to beimportant in multiple myeloma (FIG. 7C). For example, the MM.1S MYClocus contains a chromosomal rearrangement that places MYC under thecontrol of the IgH enhancers, which are highly active in the antibodyproducing plasma cells from which MM derives. The IgH-MYC locus containsa large, 40 kb super-enhancer, occupied by high levels of both BRD4 andMED1 (FIG. 7D). Super-enhancers were also found associated with the IRF4gene (FIG. 7D), which encodes a key plasma cell transcription factorfrequently deregulated in MM (Shaffer et al., 2008).

BRD4 Occupancy at Super-Enhancers is Highly Sensitive to BromodomainInhibition

Enhancers are formed through co-operative and synergistic binding ofmultiple transcription factors and coactivators (Carey, 1998; Carey etal., 1990; Giese et al., 1995; Kim and Maniatis, 1997; Thanos andManiatis, 1995). As a consequence of this binding behavior, enhancersbound by many cooperatively-interacting factors lose activity morerapidly than enhancers bound by fewer factors when the levels ofenhancer-bound factors are reduced (Giniger and Ptashne, 1988; Griggsand Johnston, 1991). The presence of super-enhancers at MYC and otherkey genes associated with multiple myeloma led us to consider thehypothesis that super-enhancers are more sensitive to reduced levels ofBRD4 than average enhancers. If super-enhancers are more sensitive toreduced levels of BRD4 than average enhancers, then super-enhancersshould experience greater loss of BRD4 than average enhancers, and genesassociated with super-enhancers might then experience a greaterreduction of transcription than genes with average enhancers.

To test this hypothesis, we first examined the effects of variousconcentrations of JQ1 on genome-wide on BRD4 occupancy (FIG. 8A). Duringthe course of the 6 hour treatments, JQ1 had little effect on MM1.S cellviability, as measured by ATP levels, while at later time points, JQ1had a dramatic antiproliferative effect (FIG. 8B). As expected, MYCprotein levels were significantly depleted by JQ1 treatment (FIG. 8C)(Delmore et al., 2011). In contrast, JQ1 did not affect BRD4 proteinlevels within cells, and did not significantly reduce ChIP efficiency(FIG. 8D). However, super-enhancers showed a greater loss of BRD4occupancy when compared to regions with average or low amounts of BRD4(FIG. 8E). The IgH enhancer was among those super-enhancers that showedsignificantly greater loss of BRD4 than typical enhancer regions withlower BRD4 occupancy, such as CD28 (FIG. 8G).

Loss of P-TEFb Accompanies BRD4 Inhibition

BRD4 recruits the active form of the positive transcription elongationfactor P-TEFb, which stimulates pause release and transcriptionelongation (Bisgrove et al., 2007; Hargreaves et al., 2009; Jang et al.,2005; Jiang et al., 1998; Wu and Chiang, 2007; Wu et al., 2003; Yang etal., 2005). We used ChIP-Seq to investigate the global occupancy ofP-TEFb in MM.1S cells and found that it generally occupies sites boundby Mediator and BRD4 (FIG. 9A). We next investigated whether the loss ofBRD4 observed with JQ1 treatment is accompanied by loss of P-TEFb atenhancers and transcription start sites. JQ1 treatment did indeed reducethe levels of P-TEFb at sites where there was a reduction in BRD4 (FIG.9B). Furthermore, P-TEFb was disproportionately lost at super-enhancerswhen compared to normal enhancers (FIG. 9C). We conclude that BETbromodomain inhibition of BRD4 leads to loss of P-TEFb at enhancers andtranscription start sites, and that the inhibition has more profoundeffects at super-enhancers than at average enhancers.

To determine whether the loss of P-TEFb results in an elongation defect,we performed ChIP-seq of RNA Polymerase II (Pol II) after JQ1 treatment.We found that JQ1 treatment led to a global defect in transcriptionalelongation, characterized by a loss of PolII in the gene body and 3′transcription termination regions (FIG. 10). Further inspection of genetracks revealed that key super-enhancer associated genes, including MYC,showed a dramatic defect in elongation (FIG. 10B). Globally,super-enhancer associated genes, had larger elongation defects inresponse to JQ1 than genes associated with normal enhancers (FIG. 10C),These results are consistent with the interpretation that genes drivenby super-enhancers show more dramatic transcriptional defects due toreduced pause release and elongation of their transcripts.

Discussion

At present, inhibitors of chromatin regulators are gaining increasedinterest as potential therapeutic agents for treating cancer. Manychromatin regulators are understood to play general roles in the controlof transcription, yet to reach significant clinical efficacy, smallmolecule inhibitors must have a selective effect on tumor cells. Severalrecent studies have shown that inhibition of the bromodomain proteinBRD4 can indeed have a highly specific effect, causing the downregulation of key tumor drivers in several cancer types. In multiplemyeloma, acute myeloid leukemia, and Burkitt's lymphoma, treatment withBET inhibitors led to a dramatic loss of MYC expression (Dawson et al.,2011; Delmore et al., 2011; Mertz et al., 2011; Zuber et al., 2011).Understanding how inhibitors of generally acting chromatin regulatorscan exert a selective effect will vastly improve our understanding ofhow these drugs should be selected for the treatment ofgenetically-defined cancers.

We have gained insight into this concept through our study ofsuper-enhancers. We have found that, across many cell types, keyregulators of cell state are associated with large, 10-40 kb enhancerdomains, characterized by disproportionately high levels of MED1 bindingand, as we have profiled in multiple myeloma, BRD4. Although thesesuper-enhancers make up only a small percentage of the total number ofenhancer regions, they account for a large fraction of total MED1 andBRD4 binding across the genome. Most significantly, we have found thatsuper-enhancers are more sensitive to perturbation than typical enhancerregions.

We found that inhibition of BRD4 led to the dramatic loss of BRD4 andCDK9 binding at super-enhancers. In multiple myeloma, super-enhancerswere associated with key oncogenic drivers, such as MYC. Disruption ofsuper-enhancers by BRD4 inhibition led to a dramatic loss of expressionof these critical tumor genes, accompanied by a potent antiproliferativeeffect.

Our results demonstrate that super-enhancers occupied by BRD4 regulatecritical oncogenic drivers multiple myeloma and show that BRD4inhibition leads to preferential disruption of these super-enhancers.This insight into the mechanism by which Brd4 inhibition causesselective loss of oncogene expression in these highly malignant bloodcancers may have implications for future drug development in oncology.Many oncogenes critical to tumor cell function are highly expressed andmay therefore be driven by super-enhancers. If so, preferentialdisruption of super-enhancer function may be a general approach toselectively inhibiting the oncogenic drivers of many tumor cells.

References

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TABLE 1 Super-enhancers from ESC. Based on NCBI Build 37 REGION_ID CHROMSTART STOP INT_STITCHED_45 chr1 13049615 13094765 INT_STITCHED_88 chr134130107 34134640 INT_STITCHED_100 chr1 36070190 36074608INT_STITCHED_101 chr1 36111164 36118698 INT_STITCHED_108 chr1 3703913937045411 INT_STITCHED_230 chr1 72260528 72261272 INT_STITCHED_237 chr172839563 72858199 INT_STITCHED_282 chr1 84857219 84887132INT_STITCHED_315 chr1 91766947 91773527 INT_STITCHED_368 chr1 120538712120545414 INT_STITCHED_372 chr1 120971968 120973737 INT_STITCHED_374chr1 121201424 121202481 INT_STITCHED_376 chr1 121295085 121296031INT_STITCHED_449 chr1 137071028 137096284 INT_STITCHED_464 chr1138586629 138593131 INT_STITCHED_466 chr1 138841643 138850970INT_STITCHED_508 chr1 154939892 154943709 INT_STITCHED_556 chr1168054897 168073079 INT_STITCHED_559 chr1 169201106 169220423INT_STITCHED_610 chr1 182818684 182819554 INT_STITCHED_611 chr1182854521 182864307 INT_STITCHED_615 chr1 183948212 183961841INT_STITCHED_746 chr2 20574602 20591747 INT_STITCHED_803 chr2 3091325730925299 INT_STITCHED_812 chr2 32008891 32030736 INT_STITCHED_817 chr233282029 33300860 INT_STITCHED_928 chr2 71488013 71494617INT_STITCHED_931 chr2 71575856 71583914 INT_STITCHED_1196 chr2 152002668152003777 INT_STITCHED_1198 chr2 152552277 152563676 INT_STITCHED_1210chr2 154242651 154254374 INT_STITCHED_1256 chr2 162856904 162860933INT_STITCHED_1257 chr2 162877048 162893236 INT_STITCHED_1279 chr2165981373 165983444 INT_STITCHED_1300 chr2 168589688 168617170INT_STITCHED_1392 chr3 9641461 9655131 INT_STITCHED_1480 chr3 3454490434553511 INT_STITCHED_1482 chr3 34633687 34660705 INT_STITCHED_1607 chr388375442 88380083 INT_STITCHED_1626 chr3 95455034 95468269INT_STITCHED_1629 chr3 96380383 96382115 INT_STITCHED_1630 chr3 9647915896484864 INT_STITCHED_1658 chr3 103008304 103019058 INT_STITCHED_1732chr3 129247012 129261362 INT_STITCHED_1744 chr3 133181431 133197648INT_STITCHED_1749 chr3 135208956 135210744 INT_STITCHED_1973 chr455469259 55491081 INT_STITCHED_2076 chr4 98507649 98514709INT_STITCHED_2152 chr4 118743867 118745786 INT_STITCHED_2175 chr4123300547 123303179 INT_STITCHED_2192 chr4 125211671 125223450INT_STITCHED_2205 chr4 126875757 126879027 INT_STITCHED_2223 chr4130178808 130180168 INT_STITCHED_2224 chr4 130195646 130196547INT_STITCHED_2265 chr4 137148873 137153839 INT_STITCHED_2268 chr4137329436 137357766 INT_STITCHED_2273 chr4 138000554 138006368INT_STITCHED_2291 chr4 140826072 140840922 INT_STITCHED_2292 chr4141120768 141126477 INT_STITCHED_2295 chr4 141616653 141627603INT_STITCHED_2297 chr4 141721916 141726166 INT_STITCHED_2317 chr4147459254 147463850 INT_STITCHED_2354 chr4 154537213 154538078INT_STITCHED_2355 chr4 154563584 154564383 INT_STITCHED_2465 chr533873714 33880481 INT_STITCHED_2510 chr5 53933177 53947327INT_STITCHED_2535 chr5 65255735 65256794 INT_STITCHED_2712 chr5113758941 113775389 INT_STITCHED_2736 chr5 116845764 116860853INT_STITCHED_2745 chr5 118884660 118896412 INT_STITCHED_2746 chr5118951444 118960269 INT_STITCHED_2752 chr5 120029649 120037063INT_STITCHED_2754 chr5 120129592 120171482 INT_STITCHED_2770 chr5123584659 123590728 INT_STITCHED_2830 chr5 135417523 135421698INT_STITCHED_3005 chr6 31834643 31852445 INT_STITCHED_3044 chr6 3937038439371286 INT_STITCHED_3045 chr6 39395571 39396779 INT_STITCHED_3120 chr664961359 64985161 INT_STITCHED_3130 chr6 67061148 67064202INT_STITCHED_3184 chr6 83839914 83844315 INT_STITCHED_3217 chr6 9164016191661247 INT_STITCHED_3342 chr6 122290093 122293017 INT_STITCHED_3347chr6 122612514 122614260 INT_STITCHED_3348 chr6 122640118 122657871INT_STITCHED_3349 chr6 122714316 122720862 INT_STITCHED_3360 chr6125383335 125398024 INT_STITCHED_3429 chr6 142458188 142461905INT_STITCHED_3437 chr6 143047309 143065758 INT_STITCHED_3450 chr6145223385 145225674 INT_STITCHED_3467 chr7 3193004 3218183INT_STITCHED_3475 chr7 4772296 4777612 INT_STITCHED_3481 chr7 1359933413600325 INT_STITCHED_3523 chr7 30982397 30983339 INT_STITCHED_3525 chr731248315 31250619 INT_STITCHED_3550 chr7 38812914 38816123INT_STITCHED_3568 chr7 52806853 52814768 INT_STITCHED_3576 chr7 5659290956604632 INT_STITCHED_3601 chr7 71092246 71102481 INT_STITCHED_3652 chr786355826 86368339 INT_STITCHED_3658 chr7 87159908 87169963INT_STITCHED_3661 chr7 87274999 87276022 INT_STITCHED_3662 chr7 8733342087345334 INT_STITCHED_3685 chr7 91027196 91051830 INT_STITCHED_3765 chr7119831735 119835688 INT_STITCHED_3856 chr7 140304156 140307245INT_STITCHED_3890 chr7 147131117 147136231 INT_STITCHED_3914 chr7152036872 152050716 INT_STITCHED_3947 chr8 12499468 12504771INT_STITCHED_4014 chr8 35023426 35027483 INT_STITCHED_4033 chr8 3760206437613850 INT_STITCHED_4034 chr8 37642521 37671979 INT_STITCHED_4046 chr844405736 44406755 INT_STITCHED_4116 chr8 74834685 74840663INT_STITCHED_4163 chr8 87174072 87174643 INT_STITCHED_4167 chr8 8799647587997654 INT_STITCHED_4179 chr8 91514813 91540176 INT_STITCHED_4190 chr893351924 93355292 INT_STITCHED_4546 chr9 56382386 56395769INT_STITCHED_4555 chr9 58119837 58128504 INT_STITCHED_4657 chr9 7820714378223442 INT_STITCHED_4748 chr9 110849422 110863371 INT_STITCHED_4766chr9 114458126 114474355 INT_STITCHED_4797 chr9 120585871 120600072INT_STITCHED_4802 chr9 121244501 121254102 INT_STITCHED_4885 chr1020802131 20830236 INT_STITCHED_4891 chr10 21546502 21549691INT_STITCHED_4893 chr10 21700576 21708946 INT_STITCHED_4954 chr1039977900 39978752 INT_STITCHED_4981 chr10 44110139 44112766INT_STITCHED_5021 chr10 59420365 59437537 INT_STITCHED_5044 chr1062346394 62361563 INT_STITCHED_5054 chr10 66380351 66383761INT_STITCHED_5059 chr10 66546199 66564235 INT_STITCHED_5091 chr1075335464 75345568 INT_STITCHED_5092 chr10 75400370 75401358INT_STITCHED_5100 chr10 76655655 76662360 INT_STITCHED_5111 chr1079508474 79515168 INT_STITCHED_5140 chr10 85002060 85006553INT_STITCHED_5325 chr11 8466451 8486876 INT_STITCHED_5331 chr11 90155379017663 INT_STITCHED_5340 chr11 12357626 12370205 INT_STITCHED_5427chr11 33427175 33451476 INT_STITCHED_5484 chr11 52173182 52184686INT_STITCHED_5499 chr11 54767341 54785832 INT_STITCHED_5533 chr1162324296 62327251 INT_STITCHED_5553 chr11 66733372 66746990INT_STITCHED_5555 chr11 66824791 66838230 INT_STITCHED_5565 chr1169517060 69522803 INT_STITCHED_ 5597 chr11 77697704 77718786INT_STITCHED_5666 chr11 88481360 88491812 INT_STITCHED_5711 chr1197517673 97524159 INT_STITCHED_5719 chr11 98823511 98826466INT_STITCHED_5741 chr11 102190649 102193692 INT_STITCHED_5752 chr11104150171 104167544 INT_STITCHED_5768 chr11 107296669 107310982INT_STITCHED_5819 chr11 116943025 116953583 INT_STITCHED_5831 chr11117833701 117838253 INT_STITCHED_5875 chr12 12790432 12795881INT_STITCHED_5876 chr12 12810177 12811020 INT_STITCHED_5880 chr1212933791 12950936 INT_STITCHED_5995 chr12 55407498 55415046INT_STITCHED_6000 chr12 56587347 56607146 INT_STITCHED_6004 chr1257385208 57400114 INT_STITCHED_6112 chr12 87807046 87820319INT_STITCHED_6113 chr12 87839385 87846192 INT_STITCHED_6118 chr1288239069 88245155 INT_STITCHED_6151 chr12 103940487 103953004INT_STITCHED_6186 chr12 111655417 111656705 INT_STITCHED_6187 chr12111709296 111710794 INT_STITCHED_6188 chr12 111725920 111743677INT_STITCHED_6460 chr13 64069823 64082322 INT_STITCHED_6544 chr1396295094 96306119 INT_STITCHED_6557 chr13 98052562 98062842INT_STITCHED_6559 chr13 98202400 98225162 INT_STITCHED_6615 chr13110418702 110442750 INT_STITCHED_6709 chr14 22293688 22308989INT_STITCHED_6789 chr14 49273113 49283200 INT_STITCHED_6815 chr1455704349 55705463 INT_STITCHED_6859 chr14 64118817 64131901INT_STITCHED_6864 chr14 65251303 65269514 INT_STITCHED_6887 chr1471022659 71035930 INT_STITCHED_6904 chr14 76894682 76915946INT_STITCHED_6906 chr14 77015215 77030315 INT_STITCHED_6957 chr1499738540 99755307 INT_STITCHED_6981 chr14 106250319 106260753INT_STITCHED_6982 chr14 106296486 106304433 INT_STITCHED_7104 chr1525654102 25704265 INT_STITCHED_7202 chr15 61918415 61924748INT_STITCHED_7248 chr15 77168852 77187251 INT_STITCHED_7285 chr1588539016 88539831 INT_STITCHED_7317 chr15 97198605 97227633INT_STITCHED_7318 chr15 97422878 97425328 INT_STITCHED_7343 chr15103349226 103353500 INT_STITCHED_7359 chr16 8758173 8779472INT_STITCHED_7434 chr16 23099373 23103471 INT_STITCHED_7452 chr1629657509 29668114 INT_STITCHED_7597 chr16 84769173 84780686INT_STITCHED_7680 chr17 10549089 10570838 INT_STITCHED_7728 chr1726631721 26648689 INT_STITCHED_7747 chr17 29209618 29218426INT_STITCHED_7752 chr17 29587776 29588942 INT_STITCHED_7767 chr1731939569 31956756 INT_STITCHED_7784 chr17 35639211 35642435INT_STITCHED_7792 chr17 37110202 37134996 INT_STITCHED_7794 chr1737209046 37217726 INT_STITCHED_7812 chr17 45593477 45596503INT_STITCHED_7822 chr17 47640414 47649043 INT_STITCHED_7876 chr1766818723 66836409 INT_STITCHED_7884 chr17 71096763 71100905INT_STITCHED_7886 chr17 71177302 71179956 INT_STITCHED_7887 chr1771213804 71222433 INT_STITCHED_7888 chr17 71241991 71250610INT_STITCHED_8114 chr18 35202713 35203454 INT_STITCHED_8124 chr1836412873 36414154 INT_STITCHED_8136 chr18 38538325 38551037INT_STITCHED_8139 chr18 38760823 38761958 INT_STITCHED_8140 chr1838788269 38796942 INT_STITCHED_8148 chr18 40467587 40468140INT_STITCHED_8209 chr18 61787544 61788400 INT_STITCHED_8260 chr1875504155 75505202 INT_STITCHED_8261 chr18 75520332 75527277INT_STITCHED_8264 chr18 75738693 75745073 INT_STITCHED_8324 chr195835881 5847014 INT_STITCHED_8378 chr19 21858770 21866770INT_STITCHED_8385 chr19 23139991 23170189 INT_STITCHED_8386 chr1923207455 23208806 INT_STITCHED_8399 chr19 25553498 25564092INT_STITCHED_8519 chr19 53523440 53535319 INT_STITCHED_8554 chrX 75789697597907 INT_STITCHED_8629 chrX 50098631 50114110

TABLE 2 Multiple Myeloma Super-enhancers. Based on Gene Build hg 18REGION_ID CHROM START STOP 3_MM1S_MED1_DMSO_2_11472_lociStitched chr2221597907 21632017 12_MM1S_MED1_DMSO_2_12661_lociStitched chr3 142561889142658635 5_MM1S_MED1_DMSO_2_11467_lociStitched chr22 21520124 215762433_MM1S_MED1_DMSO_2_15142_lociStitched chr6 7822980 786468227_MM1S_MED1_DMSO_2_15896_lociStitched chr6 108969554 10911947010_MM1S_MED1_DMSO_2_883_lociStitched chr1 117943520 11803129913_MM1S_MED1_DMSO_2_9297_lociStitched chr2 37383079 374781177_MM1S_MED1_DMSO_2_1421_lociStitched chr1 201502736 2015644746_MM1S_MED1_DMSO_2_10778_lociStitched chr20 29712568 297759674_MM1S_MED1_DMSO_2_3066_lociStitched chr11 64939923 6497993112_MM1S_MED1_DMSO_2_10818_lociStitched chr20 31862228 3193679315_MM1S_MED1_DMSO_2_19349_lociStitched chrX 130689710 1307903836_MM1S_MED1_DMSO_2_15061_lociStitched chr6 235131 282880MM1S_MED1_DMSO_2_4011 chr12 51868026 5189000813_MM1S_MED1_DMSO_2_6359_lociStitched chr16 11662193 117503995_MM1S_MED1_DMSO_2_19070_lociStitched chrX 48652795 486904489_MM1S_MED1_DMSO_2_13894_lociStitched chr4 185522607 1855862202_MM1S_MED1_DMSO_2_15298_lociStitched chr6 26263259 262819584_MM1S_MED1_DMSO_2_2709_lociStitched chr11 10280174 103017807_MM1S_MED1_DMSO_2_11528_lociStitched chr22 27516134 275559285_MM1S_MED1_DMSO_2_7255_lociStitched chr17 29712450 297455389_MM1S_MED1_DMSO_2_9712_lociStitched chr2 98426920 9849883110_MM1S_MED1_DMSO_2_5371_lociStitched chr14 90884807 909556513_MM1S_MED1_DMSO_2_7984_lociStitched chr18 9050438 90744178_MM1S_MED1_DMSO_2_16690_lociStitched chr7 55566748 556101801_MM1S_MED1_DMSO_2_935_lociStitched chr1 148122391 1481278263_MM1S_MED1_DMSO_2_3735_lociStitched chr12 12748016 127817264_MM1S_MED1_DMSO_2_2546_lociStitched chr10 125812311 1258576882_MM1S_MED1_DMSO_2_1862_lociStitched chr10 11242759 112753313_MM1S_MED1_DMSO_2_929_lociStitched chr1 147470833 147491868MM1S_MED1_DMSO_2_15293 chr6 26161696 261658913_MM1S_MED1_DMSO_2_9167_lociStitched chr2 20254183 202897761_MM1S_MED1_DMSO_2_15301_lociStitched chr6 26303073 2630949911_MM1S_MED1_DMSO_2_17447_lociStitched chr8 27264787 273401693_MM1S_MED1_DMSO_2_178_lociStitched chr1 17094196 1711397313_MM1S_MED1_DMSO_2_17882_lociStitched chr8 120985081 1210170493_MM1S_MED1_DMSO_2_1025_lociStitched chr1 153174936 1531972061_MM1S_MED1_DMSO_2_13984_lociStitched chr5 1364911 1374105MM1S_MED1_DMSO_2_15361 chr6 27964884 279720543_MM1S_MED1_DMSO_2_3071_lociStitched chr11 65020047 650354355_MM1S_MED1_DMSO_2_18418_lociStitched chr9 92710817 927461873_MM1S_MED1_DMSO_2_13885_lociStitched chr4 185421650 1854478155_MM1S_MED1_DMSO_2_9691_lociStitched chr2 96554603 9658461210_MM1S_MED1_DMSO_2_15652_lociStitched chr6 52501063 52557406MM1S_MED1_DMSO_2_7572 chr17 53760011 537730396_MM1S_MED1_DMSO_2_15868_lociStitched chr6 106637997 1066658351_MM1S_MED1_DMSO_2_15308_lociStitched chr6 26377785 263829512_MM1S_MED1_DMSO_2_7420_lociStitched chr17 38792419 388027569_MM1S_MED1_DMSO_2_14628_lociStitched chr5 131818986 1318701273_MM1S_MED1_DMSO_2_13539_lociStitched chr4 90429430 904591124_MM1S_MED1_DMSO_2_12859_lociStitched chr3 178538717 1785627224_MM1S_MED1_DMSO_2_4371_lociStitched chr12 107533824 1075604204_MM1S_MED1_DMSO_2_15314_lociStitched chr6 26449533 26475951MM1S_MED1_DMSO_2_15291 chr6 26138365 261428781_MM1S_MED1_DMSO_2_15296_lociStitched chr6 26230241 262350639_MM1S_MED1_DMSO_2_5477_lociStitched chr14 105096168 1051206882_MM1S_MED1_DMSO_2_12120_lociStitched chr3 46220865 46232443MM1S_MED1_DMSO_2_1 5292 chr6 26150596 261549522_MM1S_MED1_DMSO_2_5546_lociStitched chr15 29333964 293482401_MM1S_MED1_DMSO_2_176_lociStitched chr1 16712194 167139441_MM1S_MED1_DMSO_2_12853_lociStitched chr3 178395376 1784033531_MM1S_MED1_DMSO_2_10897_lociStitched chr20 36931952 369388622_MM1S_MED1_DMSO_2_9810_lociStitched chr2 112172513 1121825385_MM1S_MED1_DMSO_2_497_lociStitched chr1 44945879 449703117_MM1S_MED1_DMSO_2_8152_lociStitched chr18 44693277 447340291_MM1S_MED1_DMSO_2_3010_lociStitched chr11 62362909 623673382_MM1S_MED1_DMSO_2_1718_lociStitched chr1 232800286 2328162917_MM1S_MED1_DMSO_2_16140_lociStitched chr6 138287960 1383397194_MM1S_MED1_DMSO_2_16924_lociStitched chr7 101851129 1018797627_MM1S_MED1_DMSO_2_3539_lociStitched chr11 128090989 1281349465_MM1S_MED1_DMSO_2_13905_lociStitched chr4 185603808 1856340875_MM1S_MED1_DMSO_2_8400_lociStitched chr19 2546568 25797924_MM1S_MED1_DMSO_2_17232_lociStitched chr7 149685067 1497155459_MM1S_MED1_DMSO_2_6090_lociStitched chr15 88364067 884475447_MM1S_MED1_DMSO_2_5551_lociStitched chr15 29404247 294478066_MM1S_MED1_DMSO_2_908_lociStitched chr1 144138338 1441694426_MM1S_MED1_DMSO_2_2813_lociStitched chr11 22633909 226613082_MM1S_MED1_DMSO_2_11309_lociStitched chr21 40247390 402656063_MM1S_MED1_DMSO_2_11459_lociStitched chr22 21406975 214316574_MM1S_MED1_DMSO_2_4023_lociStitched chr12 52133823 521633013_MM1S_MED1_DMSO_2_6783_lociStitched chr16 78185190 781979182_MM1S_MED1_DMSO_2_2451_lociStitched chr10 112094075 1121093934_MM1S_MED1_DMSO_2_3671_lociStitched chr12 6916226 69421743_MM1S_MED1_DMSO_2_11367_lociStitched chr21 44381407 444057556_MM1S_MED1_DMSO_2_18632_lociStitched chr9 122670221 1227071395_MM1S_MED1_DMSO_2_7098_lociStitched chr17 16810645 168362435_MM1S_MED1_DMSO_2_12822_lociStitched chr3 173284485 1733095593_MM1S_MED1_DMSO_2_7795_lociStitched chr17 72647302 726723005_MM1S_MED1_DMSO_2_14194_lociStitched chr5 55473448 555005614_MM1S_MED1_DMSO_2_5843_lociStitched chr15 63374708 633853467_MM1S_MED1_DMSO_2_12921_lociStitched chr3 184711984 1847571183_MM1S_MED1_DMSO_2_13004_lociStitched chr3 195330092 1953429916_MM1S_MED1_DMSO_2_1869_lociStitched chr10 11323723 113532146_MM1S_MED1_DMSO_2_5884_lociStitched chr15 66355713 663867734_MM1S_MED1_DMSO_2_16493_lociStitched chr7 25953531 259756402_MM1S_MED1_DMSO_2_17945_lociStitched chr8 128815143 1288312623_MM1S_MED1_DMSO_2_6443_lociStitched chr16 23241697 232698551_MM1S_MED1_DMSO_2_15307_lociStitched chr6 26356880 263619494_MM1S_MED1_DMSO_2_1007_lociStitched chr1 152636911 15266053812_MM1S_MED1_DMSO_2_12617_lociStitched chr3 134643043 1347089403_MM1S_MED1_DMSO_2_1629_lociStitched chr1 224363473 2243833733_MM1S_MED1_DMSO_2_2794_lociStitched chr11 19406910 194221832_MM1S_MED1_DMSO_2_4947_lociStitched chr13 113545919 1135570863_MM1S_MED1_DMSO_2_15146_lociStitched chr6 7903492 79225242_MM1S_MED1_DMSO_2_9355_lociStitched chr2 43297983 43310825MM1S_MED1_DMSO_2_15353 chr6 27882353 278876361_MM1S_MED1_DMSO_2_117_lociStitched chr1 11889871 118931405_MM1S_MED1_DMSO_2_11097_lociStitched chr20 55481270 555092951_MM1S_MED1_DMSO_2_10440_lociStitched chr2 231437101 2314477013_MM1S_MED1_DMSO_2_340_lociStitched chr1 30988720 310059367_MM1S_MED1_DMSO_2_15801_lociStitched chr6 90115755 901427333_MM1S_MED1_DMSO_2_9401_lociStitched chr2 47380900 474044151_MM1S_MED1_DMSO_2_15359_lociStitched chr6 27939690 279440566_MM1S_MED1_DMSO_2_16939_lociStitched chr7 104350354 1043923124_MM1S_MED1_DMSO_2_14621_lociStitched chr5 131777514 1318020699_MM1S_MED1_DMSO_2_7852_lociStitched chr17 74224147 742909655_MM1S_MED1_DMSO_2_10765_lociStitched chr20 25209731 252487613_MM1S_MED1_DMSO_2_11306_lociStitched chr21 40217819 402313331_MM1S_MED1_DMSO_2_4955_lociStitched chr13 113847326 1138532799_MM1S_MED1_DMSO_2_17774_lociStitched chr8 96022708 960740481_MM1S_MED1_DMSO_2_15467_lociStitched chr6 33042969 330509912_MM1S_MED1_DMSO_2_10245_lociStitched chr2 201688028 2017012302_MM1S_MED1_DMSO_2_3620_lociStitched chr12 4086510 41002541_MM1S_MED1_DMSO_2_11604_lociStitched chr22 35056163 350614825_MM1S_MED1_DMSO_2_8117_lociStitched chr18 40542132 405603235_MM1S_MED1_DMSO_2_17304_lociStitched chr8 2016787 203776011_MM1S_MED1_DMSO_2_7624_lociStitched chr17 59486930 595367005_MM1S_MED1_DMSO_2_7793_lociStitched chr17 72590686 726182882_MM1S_MED1_DMSO_2_15176_lociStitched chr6 11937666 119442103_MM1S_MED1_DMSO_2_8375_lociStitched chr19 2032758 20491635_MM1S_MED1_DMSO_2_10377_lociStitched chr2 219449340 2194718871_MM1S_MED1_DMSO_2_18431_lociStitched chr9 92992632 92996907MM1S_MED1_DMSO_2_8809 chr19 44583388 445959316_MM1S_MED1_DMSO_2_19132_lociStitched chrX 58141354 581765682_MM1S_MED1_DMSO_2_11329_lociStitched chr21 42353240 423714853_MM1S_MED1_DMSO_2_3939_lociStitched chr12 46487401 465066362_MM1S_MED1_DMSO_2_2457_lociStitched chr10 112205500 1122154986_MM1S_MED1_DMSO_2_6074_lociStitched chr15 87434644 874757372_MM1S_MED1_DMSO_2_1061_lociStitched chr1 154382144 1543996886_MM1S_MED1_DMSO_2_14486_lociStitched chr5 109279819 1093149972_MM1S_MED1_DMSO_2_218_lociStitched chr1 23723110 237396826_MM1S_MED1_DMSO_2_11882_lociStitched chr3 5197581 52311675_MM1S_MED1_DMSO_2_8393_lociStitched chr19 2419984 24469761_MM1S_MED1_DMSO_2_11487_lociStitched chr22 22514623 225224745_MM1S_MED1_DMSO_2_11633_lociStitched chr22 35940694 359720074_MM1S_MED1_DMSO_2_13300_lociStitched chr4 39868398 398840942_MM1S_MED1_DMSO_2_15875_lociStitched chr6 106717009 1067352724_MM1S_MED1_DMSO_2_16685_lociStitched chr7 55537132 555534612_MM1S_MED1_DMSO_2_13593_lociStitched chr4 105626955 1056364981_MM1S_MED1_DMSO_2_5492_lociStitched chr14 105394828 1054006422_MM1S_MED1_DMSO_2_1032_lociStitched chr1 153236845 1532573906_MM1S_MED1_DMSO_2_6769_lociStitched chr16 77326423 773627604_MM1S_MED1_DMSO_2_15040_lociStitched chr5 180161278 1801928312_MM1S_MED1_DMSO_2_11510_lociStitched chr22 25335621 253455704_MM1S_MED1_DMSO_2_5303_lociStitched chr14 76557983 765801423_MM1S_MED1_DMSO_2_15065_lociStitched chr6 334189 3454972_MM1S_MED1_DMSO_2_10912_lociStitched chr20 40143996 401585475_MM1S_MED1_DMSO_2_6691_lociStitched chr16 66841952 668783491_MM1S_MED1_DMSO_2_7334_lociStitched chr17 35163138 351687972_MM1S_MED1_DMSO_2_18434_lociStitched chr9 93221024 932347763_MM1S_MED1_DMSO_2_8242_lociStitched chr18 58955785 589813271_MM1S_MED1_DMSO_2_13003_lociStitched chr3 195300012 1953056172_MM1S_MED1_DMSO_2_6646_lociStitched chr16 65106878 651177343_MM1S_MED1_DMSO_2_4266_lociStitched chr12 93065052 930931643_MM1S_MED1_DMSO_2_11259_lociStitched chr21 35158227 351849794_MM1S_MED1_DMSO_2_3801_lociStitched chr12 26157584 261713392_MM1S_MED1_DMSO_2_16133_lociStitched chr6 138228659 1382470513_MM1S_MED1_DMSO_2_17236_lociStitched chr7 149731864 1497498634_MM1S_MED1_DMSO_2_13002_lociStitched chr3 195258091 1952870252_MM1S_MED1_DMSO_2_953_lociStitched chr1 148798802 1488082982_MM1S_MED1_DMSO_2_1450_lociStitched chr1 203508812 2035249352_MM1S_MED1_DMSO_2_15283_lociStitched chr6 25511304 255223423_MM1S_MED1_DMSO_2_290_lociStitched chr1 26890818 269021915_MM1S_MED1_DMSO_2_7990_lociStitched chr18 9091649 91115597_MM1S_MED1_DMSO_2_18762_lociStitched chr9 133102585 1331439691_MM1S_MED1_DMSO_2_11360_lociStitched chr21 44021842 440291282_MM1S_MED1_DMSO_2_3442_lociStitched chr11 118244109 1182494983_MM1S_MED1_DMSO_2_240_lociStitched chr1 24384810 244062663_MM1S_MED1_DMSO_2_13402_lociStitched chr4 71744317 717669401_MM1S_MED1_DMSO_2_1504_lociStitched chr1 207342554 2073491642_MM1S_MED1_DMSO_2_3411_lociStitched chr11 114631374 1146416811_MM1S_MED1_DMSO_2_6445_lociStitched chr16 23321100 233269793_MM1S_MED1_DMSO_2_15550_lociStitched chr6 37230628 372524042_MM1S_MED1_DMSO_2_13986_lociStitched chr5 1388551 13992154_MM1S_MED1_DMSO_2_1441_lociStitched chr1 202729083 2027578904_MM1S_MED1_DMSO_2_1469_lociStitched chr1 204784341 2048096211_MM1S_MED1_DMSO_2_10460_lociStitched chr2 232278796 2322857742_MM1S_MED1_DMSO_2_2970_lociStitched chr11 60354930 603697713_MM1S_MED1_DMSO_2_8650_lociStitched chr19 16555465 165723889_MM1S_MED1_DMSO_2_12125_lociStitched chr3 46292850 463317091_MM1S_MED1_DMSO_2_16944_lociStitched chr7 104438848 1044439083_MM1S_MED1_DMSO_2_19007_lociStitched chrX 39838174 398544631_MM1S_MED1_DMSO_2_3626_lociStitched chr12 4247853 42572252_MM1S_MED1_DMSO_2_14483_lociStitched chr5 109219736 1092298236_MM1S_MED1_DMSO_2_12115_lociStitched chr3 46081401 461264613_MM1S_MED1_DMSO_2_11151_lociStitched chr20 61828935 618424864_MM1S_MED1_DMSO_2_5613_lociStitched chr15 38175241 381961254_MM1S_MED1_DMSO_2_13278_lociStitched chr4 37983729 379987656_MM1S_MED1_DMSO_2_5325_lociStitched chr14 81000404 810255765_MM1S_MED1_DMSO_2_8632_lociStitched chr19 16112417 161311357_MM1S_MED1_DMSO_2_6134_lociStitched chr15 91147531 911899351_MM1S_MED1_DMSO_2_7450_lociStitched chr17 40653952 406631915_MM1S_MED1_DMSO_2_1463_lociStitched chr1 204455280 2044776583_MM1S_MED1_DMSO_2_12583_lociStitched chr3 130511014 1305308741_MM1S_MED1_DMSO_2_19115_lociStitched chrX 56805175 568110382_MM1S_MED1_DMSO_2_811_lociStitched chr1 110963171 1109827991_MM1S_MED1_DMSO_2_7885_lociStitched chr17 77090061 770975393_MM1S_MED1_DMSO_2_11917_lociStitched chr3 13010123 130365595_MM1S_MED1_DMSO_2_2179_lociStitched chr10 73677336 736941263_MM1S_MED1_DMSO_2_8045_lociStitched chr18 19057373 190777071_MM1S_MED1_DMSO_2_14417_lociStitched chr5 90711139 907161882_MM1S_MED1_DMSO_2_4222_lociStitched chr12 88262387 882735971_MM1S_MED1_DMSO_2_1055_lociStitched chr1 154210608 1542188964_MM1S_MED1_DMSO_2_13888_lociStitched chr4 185476602 1855070515_MM1S_MED1_DMSO_2_13340_lociStitched chr4 47873764 479011133_MM1S_MED1_DMSO_2_902_lociStitched chr1 144093230 1441114744_MM1S_MED1_DMSO_2_4375_lociStitched chr12 107581795 1076229033_MM1S_MED1_DMSO_2_7801_lociStitched chr17 72740997 727554892_MM1S_MED1_DMSO_2_2458_lociStitched chr10 112245714 11225493411_MM1S_MED1_DMSO_2_14962_lociStitched chr5 173243900 1732894034_MM1S_MED1_DMSO_2_12387_lociStitched chr3 99962343 999788431_MM1S_MED1_DMSO_2_4479_lociStitched chr12 119212631 1192159583_MM1S_MED1_DMSO_2_15872_lociStitched chr6 106692441 1067021982_MM1S_MED1_DMSO_2_4368_lociStitched chr12 107478295 1074945483_MM1S_MED1_DMSO_2_8359_lociStitched chr19 1598817 16209295_MM1S_MED1_DMSO_2_11087_lociStitched chr20 55390112 554088655_MM1S_MED1_DMSO_2_3367_lociStitched chr11 110737473 1107654591_MM1S_MED1_DMSO_2_17494_lociStitched chr8 29685550 296904315_MM1S_MED1_DMSO_2_9346_lociStitched chr2 42179512 422107189_MM1S_MED1_DMSO_2_12137_lociStitched chr3 46384095 464135684_MM1S_MED1_DMSO_2_2241_lociStitched chr10 80670951 806904293_MM1S_MED1_DMSO_2_18151_lociStitched chr9 9596419 96057124_MM1S_MED1_DMSO_2_5951_lociStitched chr15 72850107 728655376_MM1S_MED1_DMSO_2_9773_lociStitched chr2 109176497 1092198232_MM1S_MED1_DMSO_2_11509_lociStitched chr22 25312119 253214382_MM1S_MED1_DMSO_2_13009_lociStitched chr3 195504620 1955169504_MM1S_MED1_DMSO_2_11609_lociStitched chr22 35102731 351150077_MM1S_MED1_DMSO_2_5487_lociStitched chr14 105217337 1052404891_MM1S_MED1_DMSO_2_10174_lociStitched chr2 192248312 1922536693_MM1S_MED1_DMSO_2_7356_lociStitched chr17 35720293 357371374_MM1S_MED1_DMSO_2_1849_lociStitched chr10 7553027 75752634_MM1S_MED1_DMSO_2_16642_lociStitched chr7 47479754 475048743_MM1S_MED1_DMSO_2_14100_lociStitched chr5 32607763 326259695_MM1S_MED1_DMSO_2_4572_lociStitched chr12 123957232 1239919264_MM1S_MED1_DMSO_2_2466_lociStitched chr10 112590984 1126179721_MM1S_MED1_DMSO_2_11372_lociStitched chr21 44484038 444892858_MM1S_MED1_DMSO_2_8848_lociStitched chr19 46720121 467622012_MM1S_MED1_DMSO_2_2455_lociStitched chr10 112162427 1121745743_MM1S_MED1_DMSO_2_6671_lociStitched chr16 66137623 661588133_MM1S_MED1_DMSO_2_19339_lociStitched chrX 130662431 1306735642_MM1S_MED1_DMSO_2_1640_lociStitched chr1 224906864 2249191273_MM1S_MED1_DMSO_2_7431_lociStitched chr17 39630911 396557231_MM1S_MED1_DMSO_2_8114_lociStitched chr18 40512420 405174891_MM1S_MED1_DMSO_2_14692_lociStitched chr5 138749622 1387584193_MM1S_MED1_DMSO_2_1123_lociStitched chr1 158943494 1589804884_MM1S_MED1_DMSO_2_16250_lociStitched chr6 157897104 1579137182_MM1S_MED1_DMSO_2_17101_lociStitched chr7 130440752 1304605293_MM1S_MED1_DMSO_2_9135_lociStitched chr2 11801032 118127203_MM1S_MED1_DMSO_2_10929_lociStitched chr20 42002450 420186663_MM1S_MED1_DMSO_2_1119_lociStitched chr1 158908226 1589217421_MM1S_MED1_DMSO_2_3090_lociStitched chr11 65380453 653857522_MM1S_MED1_DMSO_2_16369_lociStitched chr7 5531468 55398065_MM1S_MED1_DMSO_2_13495_lociStitched chr4 84352012 843817892_MM1S_MED1_DMSO_2_17959_lociStitched chr8 129734648 1297419731_MM1S_MED1_DMSO_2_5907_lociStitched chr15 68174162 681817684_MM1S_MED1_DMSO_2_10987_lociStitched chr20 45820090 458494232_MM1S_MED1_DMSO_2_5964_lociStitched chr15 73121792 731277992_MM1S_MED1_DMSO_2_17479_lociStitched chr8 29253249 292664445_MM1S_MED1_DMSO_2_18891_lociStitched chrX 10025533 100506772_MM1S_MED1_DMSO_2_17880_lociStitched chr8 120954422 1209696361_MM1S_MED1_DMSO_2_1813_lociStitched chr10 3814293 38188765_MM1S_MED1_DMSO_2_9497_lociStitched chr2 64716540 647482514_MM1S_MED1_DMSO_2_7748_lociStitched chr17 71366025 713873096_MM1S_MED1_DMSO_2_2420_lociStitched chr10 105235066 1052658313_MM1S_MED1_DMSO_2_7698_lociStitched chr17 68094083 681131622_MM1S_MED1_DMSO_2_18473_lociStitched chr9 97295762 973141181_MM1S_MED1_DMSO_2_7313_lociStitched chr17 34110323 341169697_MM1S_MED1_DMSO_2_4966_lociStitched chr13 114042783 1140624173_MM1S_MED1_DMSO_2_14016_lociStitched chr5 6528481 65500721_MM1S_MED1_DMSO_2_10942_lociStitched chr20 42704197 427160624_MM1S_MED1_DMSO_2_13987_lociStitched chr5 1541550 15780163_MM1S_MED1_DMSO_2_13707_lociStitched chr4 129949673 1299603742_MM1S_MED1_DMSO_2_10549_lociStitched chr2 238264326 2382779074_MM1S_MED1_DMSO_2_19321_lociStitched chrX 128720479 1287398123_MM1S_MED1_DMSO_2_9743_lociStitched chr2 105694868 1057182681_MM1S_MED1_DMSO_2_9393_lociStitched chr2 47061614 470685223_MM1S_MED1_DMSO_2_10581_lociStitched chr2 241152963 2411761725_MM1S_MED1_DMSO_2_2158_lociStitched chr10 71906942 719401733_MM1S_MED1_DMSO_2_3127_lociStitched chr11 66789831 668141092_MM1S_MED1_DMSO_2_12973_lociStitched chr3 188262377 1882749851_MM1S_MED1_DMSO_2_14147_lociStitched chr5 43072552 430796105_MM1S_MED1_DMSO_2_7714_lociStitched chr17 70249246 702791172_MM1S_MED1_DMSO_2_13272_lociStitched chr4 37805684 378123901_MM1S_MED1_DMSO_2_9642_lociStitched chr2 86073843 860821221_MM1S_MED1_DMSO_2_13666_lociStitched chr4 121888607 1218917282_MM1S_MED1_DMSO_2_6598_lociStitched chr16 55501343 555102624_MM1S_MED1_DMSO_2_12038_lociStitched chr3 39222524 392519632_MM1S_MED1_DMSO_2_1926_lociStitched chr10 15866384 158713772_MM1S_MED1_DMSO_2_5835_lociStitched chr15 62961116 629763222_MM1S_MED1_DMSO_2_11661_lociStitched chr22 37031196 370449261_MM1S_MED1_DMSO_2_2098_lociStitched chr10 63326304 633352104_MM1S_MED1_DMSO_2_3547_lociStitched chr11 128727439 1287523073_MM1S_MED1_DMSO_2_12969_lociStitched chr3 188185946 1882021482_MM1S_MED1_DMSO_2_11345_lociStitched chr21 43454397 434714573_MM1S_MED1_DMSO_2_19091_lociStitched chrX 52966103 529816423_MM1S_MED1_DMSO_2_8625_lociStitched chr19 16041917 160589193_MM1S_MED1_DMSO_2_1534_lociStitched chr1 209753487 2097687283_MM1S_MED1_DMSO_2_2769_lociStitched chr11 16923726 16943228

TABLE 3 Glioblastoma Super-Enhancers. Based on Gene Build hg18 REGION_IDCHROM START STOP 18_U87_MED1_20020_lociStitched chr3 45100470 4524352112_U87_MED1_7111_lociStitched chr12 64271490 6438049722_U87_MED1_17388_lociStitched chr2 237744314 2378961948_U87_MED1_7790_lociStitched chr12 126279637 12634465616_U87_MED1_25966_lociStitched chr6 44066339 4415388720_U87_MED1_17421_lociStitched chr2 237957090 23808675613_U87_MED1_24508_lociStitched chr5 135356769 1354408155_U87_MED1_21695_lociStitched chr3 195773224 19580195310_U87_MED1_28029_lociStitched chr7 100523787 1005710976_U87_MED1_32110_lociStitched chrX 45479800 45553892 U87_MED1_6148 chr11121548066 121570391 19_U87_MED1_15336_lociStitched chr2 4687959846970410 6_U87_MED1_28324_lociStitched chr7 130215723 13026007317_U87_MED1_28910_lociStitched chr8 23203324 232800286_U87_MED1_18087_lociStitched chr20 45376522 454240874_U87_MED1_5653_lociStitched chr11 64940094 649799489_U87_MED1_11378_lociStitched chr16 76138395 761894262_U87_MED1_19517_lociStitched chr3 4992550 50133659_U87_MED1_24067_lociStitched chr5 90604451 906466666_U87_MED1_24844_lociStitched chr5 150121686 1501558525_U87_MED1_27721_lociStitched chr7 72748931 7277483110_U87_MED1_20211_lociStitched chr3 55151891 552143476_U87_MED1_5659_lociStitched chr11 64995165 650331293_U87_MED1_19044_lociStitched chr22 28920868 289399718_U87_MED1_24834_lociStitched chr5 149974547 15002046013_U87_MED1_21089_lociStitched chr3 142532100 14262385919_U87_MED1_18679_lociStitched chr21 38534163 3864714611_U87_MED1_28206_lociStitched chr7 115938214 1160169893_U87_MED1_12623_lociStitched chr17 55257387 5527894515_U87_MED1_7069_lociStitched chr12 61372699 61474955 U87_MED1_5680chr11 65411528 65428724 U87_MED1_5682 chr11 65433153 65444824U87_MED1_19439 chr22 44836466 44869626 2_U87_MED1_3956_lociStitchedchr10 73689550 73708761 4_U87_MED1_27840_lociStitched chr7 8106742781109206 3_U87_MED1_32102_lociStitched chrX 45440741 454648415_U87_MED1_28952_lociStitched chr8 24104754 2414359521_U87_MED1_5003_lociStitched chr11 12100995 1221822211_U87_MED1_26945_lociStitched chr6 158359374 15841380010_U87_MED1_3959_lociStitched chr10 73725225 737674835_U87_MED1_3340_lociStitched chr10 17280959 173219405_U87_MED1_11362_lociStitched chr16 75864680 759008424_U87_MED1_26256_lociStitched chr6 86210398 8625012510_U87_MED1_8940_lociStitched chr14 61060615 611318167_U87_MED1_20945_lociStitched chr3 128935531 1289780897_U87_MED1_9371_lociStitched chr14 95781765 958231797_U87_MED1_17803_lociStitched chr20 29638882 296647426_U87_MED1_11435_lociStitched chr16 81214233 812492748_U87_MED1_23347_lociStitched chr5 14195028 1426199621_U87_MED1_25200_lociStitched chr5 172209298 1723163836_U87_MED1_22301_lociStitched chr4 74786901 748293478_U87_MED1_31350_lociStitched chr9 117391466 1174564362_U87_MED1_11091_lociStitched chr16 55196851 552075093_U87_MED1_16900_lociStitched chr2 207810793 20783323814_U87_MED1_4025_lociStitched chr10 76895156 769779327_U87_MED1_7635_lociStitched chr12 119145594 11918867715_U87_MED1_10048_lociStitched chr15 65153353 6523056312_U87_MED1_27257_lociStitched chr7 22565898 226240221_U87_MED1_25943_lociStitched chr6 43843596 438678544_U87_MED1_5758_lociStitched chr11 68819807 688465153_U87_MED1_22330_lociStitched chr4 75290119 7531760512_U87_MED1_9569_lociStitched chr15 30898025 3095921314_U87_MED1_25174_lociStitched chr5 172116992 17219145415_U87_MED1_29037_lociStitched chr8 28260823 283334705_U87_MED1_17455_lociStitched chr2 238996356 2390146794_U87_MED1_22339_lociStitched chr4 75448510 754805802_U87_MED1_12177_lociStitched chr17 35423480 354373029_U87_MED1_19052_lociStitched chr22 28954187 290061845_U87_MED1_20569_lociStitched chr3 100162550 1001883099_U87_MED1_23186_lociStitched chr4 189557679 18960923711_U87_MED1_31536_lociStitched chr9 129340908 1293890084_U87_MED1_23355_lociStitched chr5 14450202 14474301 U87_MED1_6146 chr11121515959 121540976 8_U87_MED1_25729_lociStitched chr6 30816520 3085896610_U87_MED1_4983_lociStitched chr11 12020084 1206915916_U87_MED1_18248_lociStitched chr20 51915427 520112992_U87_MED1_23778_lociStitched chr5 64520345 645417819_U87_MED1_7528_lociStitched chr12 110318731 1103662611_U87_MED1_7124_lociStitched chr12 64501251 6452082512_U87_MED1_12963_lociStitched chr17 73791501 7386903911_U87_MED1_19190_lociStitched chr22 35053881 3511504110_U87_MED1_21395_lociStitched chr3 171889621 1719550168_U87_MED1_1494_lociStitched chr1 94946918 9498051311_U87_MED1_18744_lociStitched chr21 41905622 419590325_U87_MED1_16409_lociStitched chr2 160767297 1608075337_U87_MED1_24782_lociStitched chr5 149368846 14942898011_U87_MED1_30075_lociStitched chr8 128970951 1290325047_U87_MED1_3670_lociStitched chr10 49466581 4951368611_U87_MED1_96_lociStitched chr1 7976379 80450807_U87_MED1_18338_lociStitched chr20 58219251 582656517_U87_MED1_24799_lociStitched chr5 149623193 14966321613_U87_MED1_31238_lociStitched chr9 113811355 11387759911_U87_MED1_4127_lociStitched chr10 80528198 805904282_U87_MED1_26448_lociStitched chr6 112461732 1124775797_U87_MED1_28103_lociStitched chr7 105771944 10580649118_U87_MED1_31033_lociStitched chr9 100772868 10088918014_U87_MED1_30675_lociStitched chr9 37974257 380601268_U87_MED1_10549_lociStitched chr15 99038601 9908999211_U87_MED1_9182_lociStitched chr14 76439874 764989697_U87_MED1_24848_lociStitched chr5 150413621 1504652466_U87_MED1_23363_lociStitched chr5 14492548 145552433_U87_MED1_13121_lociStitched chr18 3575542 359703312_U87_MED1_24585_lociStitched chr5 138995233 1390716346_U87_MED1_5284_1ociStitched chr11 35116090 351596598_U87_MED1_3521_lociStitched chr10 33284148 333452217_U87_MED1_3616_lociStitched chr10 43658240 4370852013_U87_MED1_9361_lociStitched chr14 95621243 956859624_U87_MED1_1127_lociStitched chr1 58992223 590250647_U87_MED1_12194_lociStitched chr17 35693162 357425638_U87_MED1_16879_lociStitched chr2 206252656 2063080889_U87_MED1_8736_lociStitched chr14 34901740 349464814_U87_MED1_31655_lociStitched chr9 132701910 1327278229_U87_MED1_28850_lociStitched chr8 22269372 223199006_U87_MED1_1768_lociStitched chr1 143784496 1438404294_U87_MED1_103_lociStitched chr1 8059282 80815107_U87_MED1_2854_lociStitched chr1 222980231 2230158358_U87_MED1_10117_lociStitched chr15 68542055 685887847_U87_MED1_9901_lociStitched chr15 58441168 584888323_U87_MED1_15883_lociStitched chr2 101938979 1019583913_U87_MED1_2303_lociStitched chr1 181502080 1815178733_U87_MED1_9993_lociStitched chr15 63374614 633850555_U87_MED1_4927_lociStitched chr11 10276396 103101098_U87_MED1_152_lociStitched chr1 9145323 91949087_U87_MED1_9271_lociStitched chr14 90758863 907950315_U87_MED1_292_lociStitched chr1 16147097 161663599_U87_MED1_21428_lociStitched chr3 173326232 1733832393_U87_MED1_1480_lociStitched chr1 94764000 947839458_U87_MED1_23764_lociStitched chr5 64362972 644086153_U87_MED1_29946_lociStitched chr8 123392549 1234072787_U87_MED1_12906_lociStitched chr17 72195710 722255468_U87_MED1_15990_lociStitched chr2 113267162 1133149827_U87_MED1_116_lociStitched chr1 8176575 82019705_U87_MED1_3649_lociStitched chr10 44783418 44814443 U87_MED1_12057chr17 26944014 26950786 2_U87_MED1_28437_lociStitched chr7 137207368137218994 5_U87_MED1_12467_lociStitched chr17 45458793 454982006_U87_MED1_7769_lociStitched chr12 123954906 1239917908_U87_MED1_31230_lociStitched chr9 113739183 1137772835_U87_MED1_31605_lociStitched chr9 131283335 1313119457_U87_MED1_13271_lociStitched chr18 18500819 185467856_U87_MED1_11526_lociStitched chr16 85154494 851878726_U87_MED1_10162_lociStitched chr15 72000419 720460252_U87_MED1_23340_lociStitched chr5 14085673 140956622_U87_MED1_19978_lociStitched chr3 43871898 438920963_U87_MED1_6430_lociStitched chr12 6511065 65348049_U87_MED1_19998_lociStitched chr3 45053955 450840997_U87_MED1_16195_lociStitched chr2 134284761 13431893211_U87_MED1_23940_lociStitched chr5 77835851 778853366_U87_MED1_27845_lociStitched chr7 81142953 811840644_U87_MED1_17882_lociStitched chr20 33353373 3337295510_U87_MED1_28600_lociStitched chr7 154610962 1546587535_U87_MED1_4664_lociStitched chr10 124030434 1240586914_U87_MED1_31381_lociStitched chr9 118068405 1180915013_U87_MED1_29735_lociStitched chr8 103869131 1038931375_U87_MED1_10189_lociStitched chr15 72475467 7251066610_U87_MED1_24811_lociStitched chr5 149818464 1498779857_U87_MED1_30097_lociStitched chr8 129248470 1292797333_U87_MED1_9036_lociStitched chr14 68314932 683336005_U87_MED1_20051_lociStitched chr3 46104252 461318764_U87_MED1_1138_lociStitched chr1 59085582 591226324_U87_MED1_15701_lociStitched chr2 75667467 757016384_U87_MED1_711_lociStitched chr1 33565656 335893938_U87_MED1_13446_lociStitched chr18 42508896 4255626610_U87_MED1_30394_lociStitched chr9 3846346 39078183_U87_MED1_19433_lociStitched chr22 44770633 447891448_U87_MED1_12920_lociStitched chr17 72792423 728417363_U87_MED1_29401_lociStitched chr8 62831256 628434848_U87_MED1_23950_lociStitched chr5 77897945 779477724_U87_MED1_11532_lociStitched chr16 85244100 852742823_U87_MED1_17802_lociStitched chr20 29610545 296260369_U87_MED1_7003_lociStitched chr12 55806899 558522509_U87_MED1_30667_lociStitched chr9 37919181 379595975_U87_MED1_23475_lociStitched chr5 34599305 346466199_U87_MED1_21809_lociStitched chr4 5774565 58122195_U87_MED1_2484_lociStitched chr1 199931838 1999583142_U87_MED1_18366_lociStitched chr20 60595254 606151203_U87_MED1_7103_lociStitched chr12 64204208 642187852_U87_MED1_20034_lociStitched chr3 45560540 455712713_U87_MED1_12304_lociStitched chr17 38789690 388027535_U87_MED1_22699_lociStitched chr4 123904338 1239399224_U87_MED1_9736_lociStitched chr15 43521877 435388037_U87_MED1_11894_lociStitched chr17 16864733 169084035_U87_MED1_22347_lociStitched chr4 75606560 756324027_U87_MED1_3329_lociStitched chr10 17067573 171124167_U87_MED1_11080_lociStitched chr16 54061119 540928216_U87_MED1_3934_lociStitched chr10 73013845 730356458_U87_MED1_9304_lociStitched chr14 92160646 922140118_U87_MED1_131_lociStitched chr1 8851431 88916146_U87_MED1_2968_lociStitched chr1 232801400 2328348697_U87_MED1_4111_lociStitched chr10 80355085 804084817_U87_MED1_6339_lociStitched chr12 2222492 22492994_U87_MED1_26068_lociStitched chr6 52475692 5249608110_U87_MED1_27934_lociStitched chr7 93489372 935372925_U87_MED1_3808_lociStitched chr10 64315142 643469773_U87_MED1_944_lociStitched chr1 44945138 449701743_U87_MED1_18034_lociStitched chr20 43147144 431697213_U87_MED1_325_lociStitched chr1 17094881 171137793_U87_MED1_3042_lociStitched chr1 238461272 2384896899_U87_MED1_1156_lociStitched chr1 59361216 594256696_U87_MED1_30197_lociStitched chr8 134210870 1342483213_U87_MED1_32076_lociStitched chrX 43702439 437211058_U87_MED1_5161_lociStitched chr11 27864823 279141639_U87_MED1_2459_lociStitched chr1 199680258 1997261114_U87_MED1_7235_lociStitched chr12 74697858 747177265_U87_MED1_4730_lociStitched chr10 127900118 1279329276_U87_MED1_22320_lociStitched chr4 75178405 752195735_U87_MED1_7127_lociStitched chr12 64537567 645754684_U87_MED1_17184_lociStitched chr2 226993280 2270216355_U87_MED1_16908_lociStitched chr2 207958570 2079980455_U87_MED1_15976_lociStitched chr2 113096151 1131244715_U87_MED1_19506_lociStitched chr3 4727890 47641511_U87_MED1_204_lociStitched chr1 11889850 118930623_U87_MED1_13789_lociStitched chr19 1198622 12193605_U87_MED1_4735_lociStitched chr10 128052098 1281016965_U87_MED1_31406_lociStitched chr9 122171758 1222069635_U87_MED1_9188_lociStitched chr14 76558717 765962032_U87_MED1_8973_lociStitched chr14 64612804 6461468711_U87_MED1_18186_lociStitched chr20 49369237 494193852_U87_MED1_29678_lociStitched chr8 99439088 994525264_U87_MED1_5551_lociStitched chr11 61478002 6150058310_U87_MED1_2485_lociStitched chr1 199971787 2000115276_U87_MED1_3459_lociStitched chr10 29949767 299892517_U87_MED1_17938_lociStitched chr20 36229309 362809246_U87_MED1_21893_lociStitched chr4 13498370 135444295_U87_MED1_18845_lociStitched chr21 46282572 463071125_U87_MED1_28712_lociStitched chr8 11343022 113779105_U87_MED1_11837_lociStitched chr17 13568637 135959725_U87_MED1_26443_lociStitched chr6 112399077 11244709510_U87_MED1_27794_lociStitched chr7 76875842 769333186_U87_MED1_3787_lociStitched chr10 63974312 640243953_U87_MED1_32094_lociStitched chrX 45249029 452693377_U87_MED1_2818_lociStitched chr1 221956329 2219864651_U87_MED1_7642_lociStitched chr12 119212723 1192163025_U87_MED1_15994_lociStitched chr2 113341276 1133628825_U87_MED1_20226_lociStitched chr3 55462763 554994668_U87_MED1_25357_lociStitched chr5 179683140 17971375610_U87_MED1_21194_lociStitched chr3 150772593 1508251354_U87_MED1_19522_lociStitched chr3 5033023 50542184_U87_MED1_15905_lociStitched chr2 105378414 1054013513_U87_MED1_17219_lociStitched chr2 228388427 2284081172_U87_MED1_13120_lociStitched chr18 3436350 34568964_U87_MED1_3990_lociStitched chr10 75315563 753385054_U87_MED1_11024_lociStitched chr16 49738943 497671625_U87_MED1_770_lociStitched chr1 36580667 366266854_U87_MED1_719_lociStitched chr1 33647773 336747503_U87_MED1_24071_lociStitched chr5 90698489 9071711012_U87_MED1_5468_lociStitched chr11 56798699 568502724_U87_MED1_15820_lociStitched chr2 99841615 998666594_U87_MED1_12059_lociStitched chr17 27024805 270480994_U87_MED1_19578_lociStitched chr3 10205559 102233029_U87_MED1_31314_lociStitched chr9 116906615 1169699795_U87_MED1_17381_lociStitched chr2 237695304 2377317274_U87_MED1_15023_lociStitched chr2 28463256 284864326_U87_MED1_16974_lociStitched chr2 216253277 2162870046_U87_MED1_16311_lociStitched chr2 151031128 1510618826_U87_MED1_3547_lociStitched chr10 33659030 337113775_U87_MED1_11814_lociStitched chr17 13181474 132101254_U87_MED1_28576_lociStitched chr7 151008488 1510296571_U87_MED1_13124_lociStitched chr18 3611922 36163268_U87_MED1_3534_lociStitched chr10 33444568 334941886_U87_MED1_1871_lociStitched chr1 150209432 1502414372_U87_MED1_18051_lociStitched chr20 43832868 438456223_U87_MED1_10146_lociStitched chr15 70301493 703178997_U87_MED1_2527_lociStitched chr1 201747626 20179604013_U87_MED1_24272_lociStitched chr5 112383768 1124589485_U87_MED1_26430_lociStitched chr6 112137473 1121795614_U87_MED1_13429_lociStitched chr18 41626488 416626176_U87_MED1_8590_lociStitched chr13 113882656 1139168019_U87_MED1_9551_lociStitched chr15 30738592 308023256_U87_MED1_17744_lociStitched chr20 23071349 230906276_U87_MED1_10416_lociStitched chr15 88161556 881937453_U87_MED1_5273_lociStitched chr11 35007450 350196392_U87_MED1_1556_lociStitched chr1 100859494 1008701774_U87_MED1_29271_lociStitched chr8 49481932 495081415_U87_MED1_19225_lociStitched chr22 36029312 360577155_U87_MED1_29265_lociStitched chr8 49377506 4940033510_U87_MED1_9028_lociStitched chr14 68199644 682551438_U87_MED1_12801_lociStitched chr17 67895139 679317732_U87_MED1_4101_lociStitched chr10 79683341 796945565_U87_MED1_16956_lociStitched chr2 215974532 2160118506_U87_MED1_32082_lociStitched chrX 43746648 437869324_U87_MED1_18137_lociStitched chr20 48353990 483725531_U87_MED1_5584_lociStitched chr11 62363092 623670996_U87_MED1_15922_lociStitched chr2 108226124 1082622225_U87_MED1_16864_lociStitched chr2 204370112 20438564914_U87_MED1_16427_lociStitched chr2 160916322 1609979728_U87_MED1_23630_lociStitched chr5 52329945 523699305_U87_MED1_17309_lociStitched chr2 234814049 234832679 U87_MED1_12055chr17 26929956 26934384 6_U87_MED1_23098_lociStitched chr4 182794994182847907 7_U87_MED1_2995_lociStitched chr1 233157788 2332006994_U87_MED1_4147_lociStitched chr10 80745860 8076443510_U87_MED1_25839_lociStitched chr6 35221878 352739552_U87_MED1_3179_lociStitched chr10 4794967 48088575_U87_MED1_12475_lociStitched chr17 45628535 456553443_U87_MED1_28098_lociStitched chr7 105697048 1057142771_U87_MED1_23343_lociStitched chr5 14157879 141651586_U87_MED1_20739_lociStitched chr3 113836451 1138581936_U87_MED1_2468_lociStitched chr1 199766249 1997993384_U87_MED1_4913_lociStitched chr11 9730174 97671322_U87_MED1_20084_lociStitched chr3 48567365 485795406_U87_MED1_28721_lociStitched chr8 11390711 114115345_U87_MED1_7081_lociStitched chr12 62839721 628684174_U87_MED1_23208_lociStitched chr4 190929117 1909518458_U87_MED1_15178_lociStitched chr2 37846146 378843115_U87_MED1_9939_lociStitched chr15 60965417 609809629_U87_MED1_18605_lociStitched chr21 35076849 351412367_U87_MED1_9763_lociStitched chr15 46746824 467767873_U87_MED1_27564_lociStitched chr7 45880224 458937415_U87_MED1_28912_lociStitched chr8 23294269 233257872_U87_MED1_4059_lociStitched chr10 78777531 787888695_U87_MED1_23069_lociStitched chr4 178139337 1781754853_U87_MED1_12646_lociStitched chr17 56755482 567717553_U87_MED1_16239_lociStitched chr2 143331502 1433556373_U87_MED1_29002_lociStitched chr8 26540715 265572756_U87_MED1_29815_lociStitched chr8 116499299 1165400886_U87_MED1_31373_lociStitched chr9 118032147 1180538054_U87_MED1_1780_lociStitched chr1 144138664 1441681515_U87_MED1_30166_lociStitched chr8 132922317 1329432079_U87_MED1_30246_lociStitched chr8 134963771 1350091473_U87_MED1_23445_lociStitched chr5 33334716 333575396_U87_MED1_17250_lociStitched chr2 230173938 2302071115_U87_MED1_18788_lociStitched chr21 43737139 437618426_U87_MED1_26457_lociStitched chr6 112629871 1126663126_U87_MED1_12208_lociStitched chr17 35930846 359714079_U87_MED1_28935_lociStitched chr8 23632043 236771906_U87_MED1_15610_lociStitched chr2 72004300 720319014_U87_MED1_28289_lociStitched chr7 128254122 1282698771_U87_MED1_5182_lociStitched chr11 28810629 288177092_U87_MED1_18609_lociStitched chr21 35174645 351870605_U87_MED1_28350_lociStitched chr7 130960595 1309905714_U87_MED1_22927_lociStitched chr4 158071384 1580942595_U87_MED1_4012_lociStitched chr10 76826281 768617987_U87_MED1_379_lociStitched chr1 19621887 196522246_U87_MED1_18585_lociStitched chr21 34818327 348484895_U87_MED1_904_lociStitched chr1 43160882 431820662_U87_MED1_15586_lociStitched chr2 70676208 706895472_U87_MED1_31394_lociStitched chr9 118343216 1183542001_U87_MED1_23352_lociStitched chr5 14316952 1432447210_U87_MED1_19673_lociStitched chr3 14426598 144901806_U87_MED1_4067_lociStitched chr10 78929913 789628844_U87_MED1_29950_lociStitched chr8 123509564 1235296194_U87_MED1_31528_lociStitched chr9 129297192 1293262624_U87_MED1_20561_lociStitched chr3 100091920 1001250715_U87_MED1_28581_lociStitched chr7 151055255 1510846985_U87_MED1_26426_lociStitched chr6 111980027 1120350512_U87_MED1_18956_lociStitched chr22 23149440 231632172_U87_MED1_1656_lociStitched chr1 112077002 1120887684_U87_MED1_15603_lociStitched chr2 71956835 719717563_U87_MED1_30321_lociStitched chr8 145079732 1450999913_U87_MED1_233_lociStitched chr1 12575481 126036926_U87_MED1_28749_lociStitched chr8 13254372 132799847_U87_MED1_1977_lociStitched chr1 154332675 1543671832_U87_MED1_18293_lociStitched chr20 56022630 560287839_U87_MED1_886_lociStitched chr1 41966619 420233016_U87_MED1_16981_lociStitched chr2 216300355 2163476646_U87_MED1_28927_lociStitched chr8 23451758 234817641_U87_MED1_30073_lociStitched chr8 128932139 1289370255_U87_MED1_19816_lociStitched chr3 27537533 275717767_U87_MED1_7805_lociStitched chr12 126597650 1266395725_U87_MED1_25946_lociStitched chr6 43985976 440038583_U87_MED1_28109_lociStitched chr7 105844701 1058543652_U87_MED1_9252_lociStitched chr14 89810151 898189084_U87_MED1_27267_lociStitched chr7 22723409 2273954213_U87_MED1_28793_lociStitched chr8 19068482 191312916_U87_MED1_5481_lociStitched chr11 56930199 569595612_U87_MED1_27568_lociStitched chr7 45915902 459313696_U87_MED1_4303_lociStitched chr10 95208065 952262757_U87_MED1_7132_lociStitched chr12 64596525 646397886_U87_MED1_16065_lociStitched chr2 121175738 121225198 U87_MED1_14366chr19 47304243 47311641 7_U87_MED1_18808_lociStitched chr21 4399497544024520 2_U87_MED1_5008_lociStitched chr11 12259582 122673574_U87_MED1_26112_lociStitched chr6 56306428 563443882_U87_MED1_3174_lociStitched chr10 4694138 47057917_U87_MED1_17815_lociStitched chr20 29747030 297796834_U87_MED1_8309_lociStitched chr13 79502139 795290528_U87_MED1_15724_lociStitched chr2 84968849 850071145_U87_MED1_24412_lociStitched chr5 131448786 1314687786_U87_MED1_3854_lociStitched chr10 69512331 695372554_U87_MED1_14415_lociStitched chr19 49931469 499502655_U87_MED1_14037_lociStitched chr19 13121190 131448154_U87_MED1_7978_lociStitched chr13 32722777 327589546_U87_MED1_8934_lociStitched chr14 60998858 610271731_U87_MED1_31355_lociStitched chr9 117490731 1174974524_U87_MED1_16010_lociStitched chr2 113713570 1137305978_U87_MED1_29905_lociStitched chr8 120625584 1206849521_U87_MED1_12621_lociStitched chr17 55214356 552200094_U87_MED1_18033_lociStitched chr20 43105683 431308521_U87_MED1_14566_lociStitched chr19 56760348 567709423_U87_MED1_6635_lociStitched chr12 26157496 2617982810_U87_MED1_26800_lociStitched chr6 148859778 14893000511_U87_MED1_3404_lociStitched chr10 24761351 24796199 U87_MED1_6149chr11 121571509 121574883 4_U87_MED1_30210_lociStitched chr8 134368437134385618 6_U87_MED1_1544_lociStitched chr1 99882905 999247215_U87_MED1_12392_lociStitched chr17 42688819 427273038_U87_MED1_20455_lociStitched chr3 72114549 721642679_U87_MED1_28371_lociStitched chr7 133767195 1338167934_U87_MED1_1833_lociStitched chr1 148842282 1488598881_U87_MED1_16194_lociStitched chr2 134260935 1342667645_U87_MED1_3298_lociStitched chr10 14467377 144977155_U87_MED1_19494_lociStitched chr3 4417975 44442295_U87_MED1_23525_lociStitched chr5 37806374 378296637_U87_MED1_20638_lociStitched chr3 103127775 1031670265_U87_MED1_15026_lociStitched chr2 28518271 285464475_U87_MED1_24346_lociStitched chr5 121505170 1215481411_U87_MED1_72_lociStitched chr1 7279930 72848802_U87_MED1_22344_lociStitched chr4 75583830 755908022_U87_MED1_19612_lociStitched chr3 11295015 113088745_U87_MED1_6644_lociStitched chr12 26315522 263440284_U87_MED1_18578_lociStitched chr21 34262111 342761163_U87_MED1_16960_lociStitched chr2 216100277 2161113053_U87_MED1_11901_lociStitched chr17 16984314 170013913_U87_MED1_5664_lociStitched chr11 65079515 650905364_U87_MED1_14346_lociStitched chr19 46416158 464278942_U87_MED1_24022_lociStitched chr5 86448382 864611053_U87_MED1_12721_lociStitched chr17 61694793 617090676_U87_MED1_24200_lociStitched chr5 106725129 10675398111_U87_MED1_25306_lociStitched chr5 177709555 1777488177_U87_MED1_13705_lociStitched chr18 66175119 662169885_U87_MED1_14892_lociStitched chr2 20229043 202505375_U87_MED1_358_lociStitched chr1 18060509 180786618_U87_MED1_29868_lociStitched chr8 119059307 1191018682_U87_MED1_31353_lociStitched chr9 117468982 1174769624_U87_MED1_26509_lociStitched chr6 117867944 1178801334_U87_MED1_6791_lociStitched chr12 45948501 459635071_U87_MED1_7316_lociStitched chr12 88263372 882728881_U87_MED1_28454_lociStitched chr7 139014545 1390197423_U87_MED1_29676_lociStitched chr8 99413377 994233026_U87_MED1_23651_lociStitched chr5 52728355 527629004_U87_MED1_29126_lociStitched chr8 32294513 323215471_U87_MED1_16937_lociStitched chr2 213414737 2134201895_U87_MED1_9017_lociStitched chr14 68071320 680923092_U87_MED1_7050_lociStitched chr12 61281616 612902162_U87_MED1_8479_lociStitched chr13 105600869 1056088763_U87_MED1_28991_lociStitched chr8 26361671 263783739_U87_MED1_3200_lociStitched chr10 5566940 56272263_U87_MED1_30976_lociStitched chr9 96662387 966722404_U87_MED1_29291_lociStitched chr8 49966143 500063786_U87_MED1_9106_lociStitched chr14 72173702 722039998_U87_MED1_16377_lociStitched chr2 158024751 1580543455_U87_MED1_21864_lociStitched chr4 9775313 97981684_U87_MED1_11370_lociStitched chr16 75998735 760319507_U87_MED1_15218_lociStitched chr2 39560078 395950928_U87_MED1_12696_lociStitched chr17 59756987 598129068_U87_MED1_25005_lociStitched chr5 159233084 1592733652_U87_MED1_14996_lociStitched chr2 27872924 278874111_U87_MED1_9389_lociStitched chr14 96712814 967175315_U87_MED1_27903_lociStitched chr7 92088104 921124317_U87_MED1_4679_lociStitched chr10 124239479 1242712166_U87_MED1_30005_lociStitched chr8 125807278 1258549036_U87_MED1_28456_lociStitched chr7 139069397 1390844952_U87_MED1_24155_lociStitched chr5 97670802 976793228_U87_MED1_21597_lociStitched chr3 189462566 1894985486_U87_MED1_11827_lociStitched chr17 13364635 134085797_U87_MED1_3680_lociStitched chr10 50039199 500635692_U87_MED1_19061_lociStitched chr22 29134450 291532783_U87_MED1_23518_lociStitched chr5 37750719 377607983_U87_MED1_7310_lociStitched chr12 88073577 880849691_U87_MED1_13590_lociStitched chr18 54176974 541854098_U87_MED1_8053_lociStitched chr13 42279138 423214674_U87_MED1_12184_lociStitched chr17 35505423 355241084_U87_MED1_13264_lociStitched chr18 18384075 183988742_U87_MED1_9121_lociStitched chr14 72995992 730057801_U87_MED1_318_lociStitched chr1 16712459 167137044_U87_MED1_28428_lociStitched chr7 136991184 1370324431_U87_MED1_21899_lociStitched chr4 13595377 136028466_U87_MED1_1236_lociStitched chr1 66500300 665320142_U87_MED1_23649_lociStitched chr5 52691426 527015662_U87_MED1_1146_lociStitched chr1 59278815 592850073_U87_MED1_4738_lociStitched chr10 128136077 1281484281_U87_MED1_16895_lociStitched chr2 207733794 207741019 U87_MED1_6147chr11 121541171 121547835 8_U87_MED1_18611_lociStitched chr21 3525918735300591 5_U87_MED1_21690_lociStitched chr3 195673864 1956967136_U87_MED1_24681_lociStitched chr5 142535854 1425778244_U87_MED1_25215_lociStitched chr5 172809327 1728352224_U87_MED1_14706_lociStitched chr2 9220694 92500555_U87_MED1_17204_lociStitched chr2 227939599 2279624048_U87_MED1_28194_lociStitched chr7 115849254 1158938395_U87_MED1_19772_lociStitched chr3 23665020 237033683_U87_MED1_15004_lociStitched chr2 28029902 280391534_U87_MED1_106_lociStitched chr1 8103590 81249635_U87_MED1_817_lociStitched chr1 39625358 396486926_U87_MED1_28612_lociStitched chr7 154678056 1547229457_U87_MED1_432_lociStitched chr1 21495634 215381182_U87_MED1_31267_lociStitched chr9 115421719 1154356457_U87_MED1_12576_lociStitched chr17 53290132 533263196_U87_MED1_19801_lociStitched chr3 25588763 256215093_U87_MED1_27050_lociStitched chr7 183514 1976829_U87_MED1_14480_lociStitched chr19 52127006 521868486_U87_MED1_19418_lociStitched chr22 44358335 443880852_U87_MED1_30163_lociStitched chr8 132894337 132909622 U87_MED1_14367chr19 47316482 47321267 4_U87_MED1_12273_lociStitched chr17 3792283037933478 5_U87_MED1_24689_lociStitched chr5 142592522 1426230914_U87_MED1_20269_lociStitched chr3 58004870 580210973_U87_MED1_27523_lociStitched chr7 43645607 436661451_U87_MED1_17177_lociStitched chr2 226687043 2266930052_U87_MED1_6543_lociStitched chr12 13239108 132523655_U87_MED1_17087_lociStitched chr2 220013772 2200432661_U87_MED1_30069_lociStitched chr8 128815091 1288253094_U87_MED1_29371_lociStitched chr8 59816874 598447001_U87_MED1_6537_lociStitched chr12 13141485 131482877_U87_MED1_5290_lociStitched chr11 35188819 352254402_U87_MED1_27570_lociStitched chr7 45982964 459925902_U87_MED1_14283_lociStitched chr19 43180211 431882024_U87_MED1_15878_lociStitched chr2 101801067 1018279137_U87_MED1_3818_lociStitched chr10 64389081 644358001_U87_MED1_1809_lociStitched chr1 148122343 1481275057_U87_MED1_4527_lociStitched chr10 112142380 1121770893_U87_MED1_779_lociStitched chr1 37709463 377261423_U87_MED1_28080_lociStitched chr7 104399029 1044136438_U87_MED1_18902_lociStitched chr22 19188215 192372272_U87_MED1_16544_lociStitched chr2 173720790 1737350783_U87_MED1_14022_lociStitched chr19 12749117 127665784_U87_MED1_1829_lociStitched chr1 148799756 1488195833_U87_MED1_1539_lociStitched chr1 99827254 998450833_U87_MED1_4484_lociStitched chr10 106077029 1061019485_U87_MED1_30974_lociStitched chr9 96582128 966085185_U87_MED1_8539_lociStitched chr13 109840351 1098632352_U87_MED1_21146_lociStitched chr3 147358174 1473683003_U87_MED1_13125_lociStitched chr18 3638374 36568776_U87_MED1_24416_lociStitched chr5 131578682 1316301396_U87_MED1_27818_1ociStitched chr7 80166238 801943894_U87_MED1_31023_lociStitched chr9 100662763 1006853152_U87_MED1_15229_lociStitched chr2 40176624 40187049

TABLE 4 SCLC Super-Enhancers Based on Gene Build hg 18 REGION_ID CHROMSTART STOP 1_H2171_MED1_1_1640_lociStitched chr12 6920935 69276023_H2171_MED1_1_4743_lociStitched chr20 20467079 204979127_H2171_MED1_1_1324_lociStitched chr11 44999379 450326937_H2171_MED1_1_4739_lociStitched chr20 20368291 204223373_H2171_MED1_1_4728_lociStitched chr20 20127551 201468215_H2171_MED1_1_2525_lociStitched chr14 100006544 10004108910_H2171_MED1_1_1318_lociStitched chr11 44914282 449767987_H2171_MED1_1_3367_lociStitched chr17 52974161 530207375_H2171_MED1_1_2568_lociStitched chr14 105386944 1054072204_H2171_MED1_1_2193_lociStitched chr13 70984696 709977904_H2171_MED1_1_1411_lociStitched chr11 65001189 650340885_H2171_MED1_1_2727_lociStitched chr15 67058222 670811094_H2171_MED1_1_4448_lociStitched chr2 182187487 1822168322_H2171_MED1_1_3306_lociStitched chr17 38792864 388024847_H2171_MED1_1_3117_lociStitched chr16 84027236 840777584_H2171_MED1_1_2523_lociStitched chr14 99952877 999840714_H2171_MED1_1_6398_lociStitched chr6 20798985 208174961_H2171_MED1_1_5368_lociStitched chr3 73242222 732430912_H2171_MED1_1_1409_lociStitched chr11 64938799 6495056610_H2171_MED1_1_5063_lociStitched chr22 28420926 284716603_H2171_MED1_1_1518_lociStitched chr11 110675092 1106872272_H2171_MED1_1_106_lociStitched chr1 17094879 171051117_H2171_MED1_1_370_lociStitched chr1 61124688 611643183_H2171_MED1_1_4670_lociStitched chr20 5763423 57784703_H2171_MED1_1_2458_lociStitched chr14 80493803 805241142_H2171_MED1_1_2703_lociStitched chr15 63374895 633848542_H2171_MED1_1_196_lociStitched chr1 27718317 277293481_H2171_MED1_1_1626_lociStitched chr12 1909405 19179331_H2171_MED1_1_2022_lociStitched chr12 119212791 1192161667_H2171_MED1_1_2994_lociStitched chr16 48115499 481542181_H2171_MED1_1_1385_lociStitched chr11 62364199 623670403_H2171_MED1_1_355_lociStitched chr1 60460911 604738528_H2171_MED1_1_4077_lociStitched chr2 50900527 509570403_H2171_MED1_1_4992_lociStitched chr21 45354314 453734513_H2171_MED1_1_4776_lociStitched chr20 29744744 297651111_H2171_MED1_1_86_lociStitched chr1 11890040 118929761_H2171_MED1_1_4772_lociStitched chr20 29655198 296607841_H2171_MED1_1_1806_lociStitched chr12 55914077 559243336_H2171_MED1_1_4832_lociStitched chr20 44860383 448780787_H2171_MED1_1_2352_lociStitched chr14 54625929 546538934_H2171_MED1_1_2589_lociStitched chr15 29345063 293607889_H2171_MED1_1_1076_lociStitched chr10 80658480 807126192_H2171_MED1_1_6438_lociStitched chr6 26263284 262813495_H2171_MED1_1_4748_lociStitched chr20 20518980 205542483_H2171_MED1_1_1797_lociStitched chr12 53731066 537490165_H2171_MED1_1_259_lociStitched chr1 41603873 416292602_H2171_MED1_1_4451_lociStitched chr2 182245805 1822553497_H2171_MED1_1_4066_lociStitched chr2 50831888 508740423_H2171_MED1_1_1331_lociStitched chr11 45063502 450818113_H2171_MED1_1_7960_lociStitched chr9 131283833 1313005371_H2171_MED1_1_3376_lociStitched chr17 54062985 540650192_H2171_MED1_1_3964_lociStitched chr2 8734984 87440811_H2171_MED1_1_844_lociStitched chr1 232925154 2329304962_H2171_MED1_1_3925_lociStitehed chr2 2305821 23170441_H2171_MED1_1_7716_lociStitched chr9 72222711 722263291_H2171_MED1_1_3377_lociStitched chr17 54090881 540924271_H2171_MED1_1_2879_lociStitched chr16 2456826 24628203_H2171_MED1_1_2486_lociStitched chr14 90046046 900594503_H2171_MED1_1_6363_lociStitched chr6 17580996 176008934_H2171_MED1_1_2646_lociStitched chr15 44378691 443963083_H2171_MED1_1_7981_lociStitched chr9 133669354 1336836925_H2171_MED1_1_7401_lociStitehed chr8 63107445 631355282_H2171_MED1_1_873_lociStitched chr1 241942356 2419533581_H2171_MED1_1_1285_lociStitched chr11 31851717 318551254_H2171_MED1_1_7215_lociStitched chr7 127256053 1272727114_H2171_MED1_1_2597_lociStitched chr15 29404315 294426872_H2171_MED1_1_4761_lociStitched chr20 24646465 246522043_H2171_MED1_1_4781_lociStitched chr20 30575765 305891402_H2171_MED1_1_3111_lociStitched chr16 83975326 839895314_H2171_MED1_1_488_lociStitched chr1 116596631 1166131223_H2171_MED1_1_7399_lociStitched chr8 62993333 630239124_H2171_MED1_1_4597_lociStitched chr2 232245138 2322579675_H2171_MED1_1_1703_lociStitehed chr12 28479296 284979044_H2171_MED1_1_8014_lociStitched chr9 136814438 1368333292_H2171_MED1_1_2533_lociStitched chr14 100108519 1001266735_H2171_MED1_1_6671_lociStitched chr6 112078789 1120911563_H2171_MED1_1_6930_lociStitched chr7 31684707 316992722_H2171_MED1_1_277_lociStitched chr1 44959548 449699241_H2171_MED1_1_4770_lociStitched chr20 29623515 296260663_H2171_MED1_1_3229_lociStitched chr17 18824205 188385085_H2171_MED1_1_7373_lociStitched chr8 53305190 533307602_H2171_MED1_1_4445_lociStitched chr2 182146929 1821606145_H2171_MED1_1_6182_lociStitched chr5 142369672 1423975493_H2171_MED1_1_3109_lociStitched chr16 83939477 839571331_H2171_MED1_1_6436_lociStitched chr6 26230266 262349692_H2171_MED1_1_5573_lociStitched chr3 171666644 1716726014_H2171_MED1_1_7990_lociStitched chr9 133870805 1338894097_H2171_MED1_1_324_lociStitched chr1 54535842 545958843_H2171_MED1_1_4733_lociStitched chr20 20330857 203400221_H2171_MED1_1_1286_lociStitched chr11 31970692 319751433_H2171_MED1_1_6477_lociStitched chr6 33819339 338288494_H2171_MED1_1_5144_lociStitched chr22 41520431 415408322_H2171_MED1_1_5576_lociStitched chr3 171727766 1717340923_H2171_MED1_1_7552_lociStitched chr8 125856085 1258721493_H2171_MED1_1_7535_lociStitched chr8 123754555 1237659253_H2171_MED1_1_5948_lociStitched chr5 14793111 148101192_H2171_MED1_1_5868_lociStitched chr4 141377946 1413944032_H2171_MED1_1_1526_lociStitched chr11 110802193 1108137151_H2171_MED1_1_3506_lociStitched chr17 75396929 754024144_H2171_MED1_1_4283_lociStitched chr2 134996095 1350110033_H2171_MED1_1_6663_lociStitched chr6 111978600 1119957523_H2171_MED1_1_858_lociStitched chr1 235546011 2355566311_H2171_MED1_1_3207_lociStitched chr17 8016708 80185894_H2171_MED1_1_303_lociStitched chr1 53346865 533791753_H2171_MED1_1_6854_lociStitched chr7 3273583 32824594_H2171_MED1_1_2201_lociStitched chr13 71269244 712876352_H2171_MED1_1_5514_lociStitched chr3 141542495 1415477051_H2171_MED1_1_102_lociStitched chr1 16712502 167138361_H2171_MED1_1_3304_lociStitched chr17 38747760 387495884_H2171_MED1_1_3851_lociStitched chr19 43240289 432574083_H2171_MED1_1_7984_lociStitched chr9 133750060 1337672552_H2171_MED1_1_2593_lociStitched chr15 29374833 293820924_H2171_MED1_1_1632_lociStitched chr12 3191844 32086893_H2171_MED1_1_613_lociStitched chr1 181446125 1814558121_H2171_MED1_1_515_lociStitched chr1 147489769 1474917151_H2171_MED1_1_7564_lociStitched chr8 127859208 1278717212_H2171_MED1_1_4141_lociStitched chr2 70212694 702245251_H2171_MED1_1_3928_lociStitched chr2 2827367 28306923_H2171_MED1_1_3104_lociStitched chr16 83865721 838790793_H2171_MED1_1_7998_lociStitched chr9 134078841 1340970473_H2171_MED1_1_7465_lociStitched chr8 93687205 936939133_H2171_MED1_1_2733_lociStitched chr15 67212499 672375564_H2171_MED1_1_2715_lociStitched chr15 64230622 642502112_H2171_MED1_1_1770_lociStitched chr12 48729670 487339845_H2171_MED1_1_5251_lociStitched chr3 16817691 168412852_H2171_MED1_1_8026_lociStitched chr9 137161098 1371702114_H2171_MED1_1_5775_lociStitched chr4 80519421 805367213_H2171_MED1_1_4835_lociStitched chr20 45030700 450425382_H2171_MED1_1_5461_lociStitched chr3 127738334 1277473821_H2171_MED1_1_3360_lociStitched chr17 52949374 529523303_H2171_MED1_1_4792_lociStitched chr20 31606072 316290763_H2171_MED1_1_5099_lociStitched chr22 36154719 361759743_H2171_MED1_1_3453_lociStitched chr17 69839454 698506582_H2171_MED1_1_606_lociStitched chr1 180846204 1808555653_H2171_MED1_1_2977_lociStitched chr16 47549993 475645435_H2171_MED1_1_5509_lociStitched chr3 141387464 1414088274_H2171_MED1_1_6832_lociStitched chr7 1281279 13057383_H2171_MED1_1_7419_1ociStitched chr8 64128807 641522943_H2171_MED1_1_392_lociStitched chr1 67883445 678918313_H2171_MED1_1_7851_lociStitched chr9 111073312 1110815181_H2171_MED1_1_3482_lociStitched chr17 73307672 733119621_H2171_MED1_1_677_lociStitched chr1 200341028 2003449534_H2171_MED1_1_2770_lociStitched chr15 72303789 723256731_H2171_MED1_1_1390_lociStitched chr11 63440196 634453565_H2171_MED1_1_5682_lociStitched chr4 8071726 80981322_H2171_MED1_1_358_lociStitched chr1 60514461 605205112_H2171_MED1_1_8028_lociStitched chr9 137386784 1373964434_H2171_MED1_1_4955_lociStitched chr21 38139981 381651656_H2171_MED1_1_1142_lociStitched chr10 112592513 1126151094_H2171_MED1_1_672_lociStitched chr1 200253434 2002744072_H2171_MED1_1_1016_lociStitched chr10 73690165 737064283_H2171_MED1_1_5762_lociStitehed chr4 80338728 803524942_H2171_MED1_1_6852_lociStitched chr7 3187356 31958402_H2171_MED1_1_4985_lociStitched chr21 44518734 445248113_H2171_MED1_1_4943_lociStitched chr21 33430682 334473081_H2171_MED1_1_3368_lociStitched chr17 53035354 530406444_H2171_MED1_1_2427_lociStitched chr14 76442631 764617004_H2171_MED1_1_4558_lociStitched chr2 217169924 2171961873_H2171_MED1_1_7744_lociStitched chr9 85109090 851229795_H2171_MED1_1_1691_lociStitched chr12 28264210 282815143_H2171_MED1_1_2892_lociStitched chr16 11047692 110598492_H2171_MED1_1_4899_lociStitched chr20 60880400 608853601_H2171_MED1_1_7947_lociStitched chr9 129461873 1294645182_H2171_MED1_1_674_lociStitched chr1 200292057 2003068462_H2171_MED1_1_2203_lociStitched chr13 71325559 713384292_H2171_MED1_1_3186_lociStitched chr17 3729010 37419422_H2171_MED1_1_2909_lociStitched chr16 11781980 117947512_H2171_MED1_1_4766_lociStitched chr20 25613739 256205303_H2171_MED1_1_2897_lociStitched chr16 11144367 111544521_H2171_MED1_1_5910_lociStitched chr5 451422 4542664_H2171_MED1_1_7415_lociStitched chr8 63776687 638061551_H2171_MED1_1_1453_lociStitched chr11 78328712 783312623_H2171_MED1_1_6678_lociStitched chr6 112348676 1123562732_H2171_MED1_1_1765_lociStitched chr12 48546951 485551511_H2171_MED1_1_2433_lociStitched chr14 76568042 765707771_H2171_MED1_1_7993_lociStitched chr9 133904790 1339088372_H2171_MED1_1_3164_lociStitched chr17 1642357 16478884_H2171_MED1_1_1653_lociStitched chr12 8501768 85232912_H2171_MED1_1_8030_lociStitched chr9 138135585 1381420784_H2171_MED1_1_6697_lociStitched chr6 114858043 1148744062_H2171_MED1_1_2731_lociStitched chr15 67146253 671531081_H2171_MED1_1_676_lociStitched chr1 200320223 2003234633_H2171_MED1_1_7106_lociStitched chr7 90891248 909046463_H2171_MED1_1_1282_lociStitched chr11 31605924 316227631_H2171_MED1_1_6439_lociStitched chr6 26304930 263088403_H2171_MED1_1_6542_lociStitched chr6 43874271 438916272_H2171_MED1_1_362_lociStitched chr1 61095855 611054843_H2171_MED1_1_4288_lociStitched chr2 135067807 1350786751_H2171_MED1_1_2649_lociStitched chr15 44422399 444252934_H2171_MED1_1_1859_lociStitched chr12 74259275 742828032_H2171_MED1_1_6347_lociStitched chr6 15255876 152615983_H2171_MED1_1_3953_lociStitched chr2 7321393 73283141_H2171_MED1_1_3540_lociStitched chr17 77837805 778400242_H2171_MED1_1_6860_lociStitched chr7 5429395 54461662_H2171_MED1_1_3999_lociStitched chr2 23567475 235728881_H2171_MED1_1_5475_lociStitched chr3 130776820 1307814691_H2171_MED1_1_4528_lociStitched chr2 207882381 2078851582_H2171_MED1_1_842_lociStitched chr1 232900985 2329036732_H2171_MED1_1_3986_lociStitched chr2 20412482 204200652_H2171_MED1_1_3326_lociStitched chr17 43434796 434478303_H2171_MED1_1_5929_lociStitched chr5 8720521 87375552_H2171_MED1_1_4569_lociStitched chr2 218968606 2189809383_H2171_MED1_1_890_lociStitched chr10 1486532 14938971_H2171_MED1_1_7959_lociStitched chr9 131260227 1312635913_H2171_MED1_1_2166_lociStitched chr13 58914764 589256841_H2171_MED1_1_8018_lociStitched chr9 136926589 1369302264_H2171_MED1_1_3837_lociStitched chr19 40215562 402399462_H2171_MED1_1_6546_lociStitehed chr6 43906068 439126571_H2171_MED1_1_331_lociStitched chr1 54796212 547990142_H2171_MED1_1_1637_lociStitched chr12 3677173 36876802_H2171_MED1_1_4907_lociStitched chr20 61600274 61610725]_H2171_MED1_1_4771_lociStitched chr20 29638614 296402391_H2171_MED1_1_6475_lociStitched chr6 33043033 330487204_H2171_MED1_1_6405_lociStitched chr6 21296380 213107342_H2171_MED1_1_5758_lociStitched chr4 80183595 801918611_H2171_MED1_1_7556_lociStitched chr8 126466494 1264688432_H2171_MED1_1_4002_lociStitched chr2 23606108 236132902_H2171_MED1_1_2058_lociStitched chr12 123805217 1238102132_H2171_MED1_1_849_lociStitched chr1 233312827 2333214593_H2171_MED1_1_3899_lociStitched chr19 53527025 535468262_H2171_MED1_1_395_lociStitched chr1 67912539 679166654_H2171_MED1_1_6138_lociStitched chr5 134851883 1348659952_H2171_MED1_1_4060_lociStitched chr2 50612904 506178302_H2171_MED1_1_4365_lociStitched chr2 155710278 1557200402_H2171_MED1_1_7889_lociStitched chr9 119201834 1192072121_H2171_MED1_1_7587_lociStitched chr8 134455564 1344587753_H2171_MED1_1_2601_lociStitched chr15 29458295 294787933_H2171_MED1_1_1260_lociStitched chr11 22313265 223226003_H2171_MED1_1_5218_lociStitched chr3 10469904 104871093_H2171_MED1_1_6827_lociStitched chr7 1204071 12179695_H2171_MED1_1_2046_lociStitehed chr12 123556670 1235833122_H2171_MED1_1_193_lociStitched chr1 27053645 270654302_H2171_MED1_1_4672_lociStitched chr20 5798497 58083344_H2171_MED1_1_1335_lociStitched chr11 45327480 453505832_H2171_MED1_1_3922_lociStitched chr2 1989631 20005223_H2171_MED1_1_1783_lociStitched chr12 51552711 515605571_H2171_MED1_1_4572_lociStitched chr2 219568763 2195742661_H2171_MED1_1_8038_lociStitched chr9 138634973 1386401382_H2171_MED1_1_450_lociStitched chr1 107836814 1078407281_H2171_MED1_1_6340_lociStitched chr6 15092300 150953111_H2171_MED1_1_3904_lociStitched chr19 53829063 538338375_H2171_MED1_1_5224_lociStitched chr3 11299162 113240952_H2171_MED1_1_5986_lociStitched chr5 35395346 354068913_H2171_MED1_1_1693_lociStitched chr12 28296478 283095751_H2171_MED1_1_5922_lociStitched chr5 3542098 35463823_H2171_MED1_1_2604_lociStitched chr15 29544679 295608272_H2171_MED1_1_2254_lociStitched chr13 99297247 993065034_H2171_MED1_1_2041_lociStitched chr12 123445277 1234747142_H2171_MED1_1_2141_lociStitched chr13 52466506 524786016_H2171_MED1_1_817_lociStitched chr1 230727775 2307582553_H2171_MED1_1_2411_lociStitched chr14 73925775 739399831_H2171_MED1_1_6346_lociStitched chr6 15239481 152420622_H2171_MED1_1_31_lociStitched chr1 6252175 62615232_H2171_MED1_1_6334_lociStitched chr6 14651962 146584901_H2171_MED1_1_10_lociStitched chr1 1355263 13601551_H2171_MED1_1_534_lociStitched chr1 153237908 1532442711_H2171_MED1_1_3988_lociStitched chr2 20600392 206054691_H2171_MED1_1_5909_lociStitched chr5 423764 4283581_H2171_MED1_1_4170_lociStitched chr2 86115761 861184111_H2171_MED1_1_7411_lociStitched chr8 63579148 635861163_H2171_MED1_1_2624_lociStitched chr15 37633152 376386291_H2171_MED1_1_5911_lociStitched chr5 695232 6978603_H2171_MED1_1_1253_lociStitched chr11 19713467 197247533_H2171_MED1_1_2541_lociStitched chr14 100247113 1002542432_H2171_MED1_1_7301_lociStitched chr7 157275487 1572821121_H2171_MED1_1_5103_lociStitched chr22 36214156 362181823_H2171_MED1_1_3372_lociStitched chr17 53332524 533503282_H2171_MED1_1_2657_lociStitched chr15 45586454 455939692_H2171_MED1_1_4694_lociStitched chr20 12456692 124650192_H2171_MED1_1_6684_lociStitched chr6 112571616 1125828171_H2171_MED1_1_3996_lociStitched chr2 22829298 228318112_H2171_MED1_1_8040_lociStitched chr9 138704382 1387111691_H2171_MED1_1_5920_lociStitched chr5 3445869 34491961_H2171_MED1_1_5760_lociStitehed chr4 80303607 80306058

1.-26. (canceled)
 27. A cell transfected with a nucleic acid constructcomprising a super-enhancer, the super-enhancer comprising: a genomicregion of deoxyribonucleic acid (DNA) that contains at least twoenhancers, wherein the genomic region is occupied when present within acell by an order of magnitude more transcriptional coactivator orchromatin regulator than the average single enhancer within the cell,wherein upon transfection of the cell with the nucleic acid constructendogenous transcriptional coactivators and chromatin regulators withinthe cell co-occupy the enhancers and the active transcription startsites of the target gene to stimulate high levels of expression of thetarget gene within the cell.
 28. The cell of claim 27, wherein the cellis a mammalian cell.
 29. The cell of claim 27, wherein the cell is ahuman cell.
 30. The cell of claim 27, wherein the cell is an embryonicstem cell or embryonic stem cell-like cell.
 31. The cell of claim 27,wherein the cell is a muscle cell.
 32. The cell of claim 31, wherein themuscle cell is a myotube.
 33. The cell of claim 27, wherein the cell isa B cell.
 34. The cell of claim 33, wherein the B cell is a Pro-B cell.35. A method of increasing the level of expression of a target gene in acell, comprising transfecting a cell under conditions suitable forexpression of the target gene with a nucleic acid expression constructcomprising a nucleic acid sequence encoding the target gene operativelylinked to a super-enhancer, wherein upon transfection of the cellendogenous transcriptional coactivators and chromatin regulators withinthe cell co-occupy enhancers clustered within the super-enhancer by anorder of magnitude more than the average single enhancer within the celland active transcription start sites of the target gene to increase thelevel of expression of the target gene within the cell.
 36. The methodof claim 35, wherein the level of expression of the target gene isincreased 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, or more within thecell.
 37. A kit for increasing the expression of a target gene in acell, comprising: (a) a nucleic acid construct comprising an artificialsuper-enhancer operatively linked to the target gene; (b) a populationof cells suitable for expression of said target gene; and (c) a reagentfor transfecting said population of cells with said nucleic acidconstruct.
 38. A method of identifying a super-enhancer in a cell,comprising: (a) identifying a genomic region of DNA within said cellcharacterized by a cluster of enhancers each of which bind a cognatetranscription factor capable of interacting with Mediator to stimulatetranscription of the target gene within said cell; (b) measuring in theidentified genomic region a level of Mediator; and (c) identifying thegenomic region as a super-enhancer if the level of Mediator identifiedin the genomic region is an order of magnitude greater than the level ofMediator occupying the average single enhancer. 39.-40. (canceled) 41.The method of claim 38, wherein the super-enhancer is identified byperforming chromatin immunoprecipitation high-throughput sequencing(ChIP-Seq). 42.-50. (canceled)
 51. A method of treating a proliferativedisorder in a patient in need of such treatment, said proliferativedisorder characterized by an oncogene-associated super-enhancer occupiedby more Mediator or BRD4 than an average single enhancer, comprisingadministering to the patient an effective amount of an agent thatdisrupts the function of the oncogene-associated super-enhancer, therebyselectively inhibiting proliferation of the oncogene in the patient. 52.The method of claim 51, wherein the proliferative disorder is ahematological malignancy.
 53. The method of claim 51, wherein theproliferative disorder is selected from the group consisting of acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), hairycell leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneousT-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), Mantle celllymphoma, B-cell lymphoma, acute lymphoblastic T cell leukemia (T-ALL),acute promyelocytic leukemia, and multiple myeloma.
 54. The method ofclaim 51, wherein the agent is a BRD4 inhibitor.
 55. The method of claim51, wherein the agent is JQ1.
 56. (canceled)
 57. A method of treatingmultiple myeloma involving an IGH-MYC locus that results in aberrantexpression of oncogene c-Myc, comprising administering to a patient inneed of such treatment an effective amount of an agent that decreasesoccupancy levels of BRD4 and MED1 at a super-enhancer region associatedwith the IGH-MYC locus, wherein decreased occupancy levels of BRD4 andMED1 at the super-enhancer disrupt function of the super-enhancerthereby decreasing aberrant expression of oncogene c-Myc such that themultiple myeloma is treated.
 58. The method of claim 57, wherein theagent is JQ1 or iBET.
 59. (canceled)
 60. A method of identifying anagent that disrupts a super-enhancer comprising: (a) transfecting a cellwith a super-enhancer operably linked to a reporter construct comprisinga reporter gene under conditions suitable for the super-enhancer todrive high levels of expression of the reporter gene; (b) contacting thecell with a test agent; (c) and measuring the level of expression of thereporter gene, wherein decreased expression of the reporter gene in thepresence of the test agent indicates that the test agent is as an agentthat disrupts the super-enhancer.
 61. The method of claim 60, whereinthe super-enhancer is naturally associated with a gene of interest,wherein the gene of interest is optionally a disease-associated gene,optionally an oncogene.
 62. The method of claim 60 wherein expression ismeasured at least in part by measuring the level of a gene productencoded by the gene or by measuring activity of a gene product encodedby the gene.
 63. The method of claim 62 wherein a gene product is mRNAor polypeptide encoded by the gene.
 64. The method of claim 61 whereinexpression is measured at least in part by measuring the level of a geneproduct encoded by the gene or by measuring activity of a gene productencoded by the gene.